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Full text of "The microscope; an introduction to microscopic methods and to histology"

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THE METRIC SYSTEM 




10 CENTIMETER RULE 

The upper edge is in millimeters, the lower in centimeters, and half centimeters. 



THE METER FOR 
LENGTH. . . 



UNITS. The most commonly used divisions and multiples. 

(Centimeter (cm.), o.oi Meter; Millimeter (mm.), o.ooi Meter: Micron (u.), 
•< o.ooi Millimeter ; the Micron is the unit in Micrometry (J 166). 
{^Kilometer, iooo Meters ; used in measuring roads and other long distances. 
the gram for \ Milligram (mg.), o.ooi Gram. 

■weight. . . ( Kilogram, iooo Grams, used for ordinary masses, like groceries, etc. 
the liter for I Cubic Centimeter (cc), o.ooi Liter. This is more common than the correct 
capacity. . \ form, Milliliter. 
Divisions of the Units are indicated by the Latin prefixes : deci, o.i ; centi, o.oi ; Milli, 
o.ooi ; Mia-o, one millionth (o.oooooi) of any unit. 

Multiples are designated by the Greek prefixes : deka, 10 times ; hecto, ioo times ; kilo, iooo 
timse ; myria. 10.000 times : Meza. one million (1,000,000) times any unit. 



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Farenheit to Centigrade, F.= — 40 (— 40 — 32°)= — 72 , whence — 72°Xf 
= — 40 C. 

Address of American Opticians : For the price of microscopes and microscopical supplies the 
student is advised to obtain a catalog of one or more of the opticians. Nearly all of them import 
foreign apparatus. For all institutions entitled to duty free importation, American microscopes 
are sold at duty free rates. For the foreign opticians see the table of tube-length p. 14. 

The Bausch and Lomb Optical Co., New York, Rochester, and Chicago. 

Eimer and Amend, 205-211 3d Ave., New York. 

The Franklin Laboratory Supply Co , Harcourt St., Boston, Mass. 

The Gundlach Optical Company, Rochester, N. Y. 

Win. Krafft (Representative of Leitz in America), 4" West 59th St., New York. 

Edward Pennock 3609 Woodland Ave., Philadelphia, Pa. 

Oueen & Company, 1010 Chestnut St., Philadelphia, Pa. 

Richards & Co., .— 12 East iSth St., New York, and 108 Lake St., Chicago, Ills. 

Spencer Lens Company, 3 6 7~373 Seventh St., Buffalo, N. Y. 

Williams, Brown & Earle, 918 Chestnut St., Philadelphia, Pa. 

G. S. Woolman (Queen & Co. in New York), 116 Fulton St., New York. 

Joseph Zentma5'er, 226-228 South 15th St., Philadelphia, Pa. 

Besides the names here given, nearly every large city has one or more dealers in microscopes 
and microscope supplies. 



THE MICROSCOPE 

AN 
INTRODUCTION TO MICROSCOPIC 
METHODS AND TO HISTOLOGY 



BY SIMON HENRY GAGE 

/I i 

PROFESSOR OF MICROSCOPY, HISTOL- 
OGY AND EMBRYOLOGY IN CORNELL 
UNIVERSITY, AND THE NEW YORK 
STATE VETERINARY COLLEGE. 



£~ 



EIGHTH 




EDITION 



REVISED, ENLARGED AND ILLUSTRATED BY OVER 
TWO HUNDRED FIGURES 

COMSTOCK PUBLISHING COMPANY 

ITHACA, NEW YORK 
1901 



4 i ■„ o ^ 




Copyright, 1901. 

By Simon Henry Gage. 

All Rights Reserved. 




Press of 

The Ithaca Journal 

Ithaca, N. Y. 






ft It 




PREFACE TO THE EIGHTH EDITION 



AS the preface to the sixth edition of this work expresses accurately what 
should be said to-day it is appended : 

1 'The rapid advance in microscopical knowledge, and the great strides in the sci- 
ences employing the microscope as an indispensable tool, have reacted upon the 
microscope itself, and never before were microscopes so excellent, convenient and 
cheap. Indeed, the financial reason for not possessing a microscope can no longer 
be urged by any high school or academy, or by any person whose profession de- 
mands it. 

Naturally, to get the greatest good from instruments, tools, or machines of any 
kind, the one who uses them must understand the principles upon which their 
action depends, their possibilities and limitations. 

That the student may acquire a just comprehension of some of the fundamental 
principles of the microscope, and gain a working acquaintance with it and its ap- 
plications, this book has been prepared. It is a growth of the laboratory, and has 
been modified from time to time to keep pace with optical improvements and ad- 
vancing knowledge." 

In rewriting this edition the different chapters have been recast, new figures 
added and in most cases the matter considerably increased. A new chapter has 
been added upon class demonstrations. The general availability of the constant 
electric current, and the improvement in apparatus have made micro-projection 
practicable and satisfactory. It has served the writer so well in his teaching of 
histology and embryology that it seemed worth while to give the benefit of his ex- 
perience to his fellow workers. 

It is hoped that the book as it now stands will serve more completely than 
ever before the needs of the class-room and of the laboratory. 

"Simply reading a work on the microscope, and looking a few times into an 
instrument completely adjusted by another, is of very little value in giving real 
knowledge. In order that the knowledge shall be made alive, it must become a 
part of the student's experience by actual experiments carried out by the student 
himself. Consequently, exercises illustrating the principles of the microscope 
and the methods of its employment have been made an integral part of the work. 

"In considering the real greatness of the microscope, and the truly splendid 
service it has rendered, the fact has not been lost sight of that the microscope is, 
after all, only an aid to the eye of the observer, only a means of getting a larger 
image on the retina than would be possible without it ; but the appreciation of 



iv PREFACE 

the retinal image, whether it is made with or without the aid of a microscope, 
must always depend upon the character and training of the seeing and appreciat- 
ing brain behind the eye. The microscope simply aids the eye in furnishing raw 
material, so to speak, for the brain to work upon. (From 3d ed. ) 

Grateful acknowledgment is made to the opticians and instrument makers 
for the loan of cuts and for courteous and complete answers to numerous question ; 
to the directors of laboratories in different parts of the country, to his colleagues 
in the departments of Physics, Chemistry and Electrical Engineering in Cornell 
University; and finally to his pupils past and present wbo have given their sup- 
port and encouragement. 

In closing I would like to urge those who are interested in Microscopy to 
take same microscopical journal, and if possible to become a member of some 
microscopical club or society. One can do very little alone, but by helping others 
and being helped in return, the workers in any field of human endeavor can 
accomplish great things. 

SIMON HENRY GAGE, 
Cornell University, 

October /, /go/. Ithaca, N. Y., U. S. A. 



CONTENTS 



CHAPTER I 

PAGE 

§ I_ 59 — The Microscope and its parts I- 33 

CHAPTER II 

? 60-128 — Lighting and Focusing; Manipulation of Dry, Adjustable 
and Immersion Objectives ; Care of the Microscope and of 
' theEyes. Laboratory Microscopes 34-89 

CHAPTER III 
I 129-153 — Interpretation of Appearances 90-102 

CHAPTER IV 
I 154-176 — Magnification and Micrometry 103-121 

CHAPTER V 
\ 177-187 — Drawing with the Microscope 122-133 

CHAPTER VI 

§ 188-233 — The Microspectroscope and Polariscope ; Micro- Chemistry ; 

Textile Fibers and Food Products ; Micro-Metallography 134-160 

CHAPTER VII 

I 234-335 — Slides and Cover-Glasses ; Mounting ; Isolation ; Sectioning 
by the Collodion and the Paraffin Methods ; Serial Sections ; 
Labeling and Storing Microscopical Preparations ; Reagents 
and their Preparation . 161-204 

CHAPTER VIII 

'i 336-39 2 — Photographing objects with a Vertical Camera ; Photograph- 
ing Large Transparent Objects ; Photographing with a Mi- 
croscope ; (A) Transparent Objects ; (B) Opaque Objects, 
and the Surfaces of Metals and Alloys ; Enlarging Nega- 
tives ; Photographing Petri Dishes and Culture Tubes 205-242 

CHAPTER IX 

\ 393-426 — Class-Room Demonstrations with the Microscope ; With the 

Projection Microscope; with the Episcope 243.267 

CHAPTER X 

\ 427-438 — The Abbe Test-Plate and Apertometer ; Equivalent Focus of 
Objectives and Oculars ; Drawings for Photo-Engravings ; 
Wax Models ; Some Apparatus for Imbedding and Section- 
ing 268-281 

BOOKS AND PERIODICALS 282-288 

INDEX 289 




THE MICROSCOPE IN SECTION 



Huygenian ocular (see p. 102 for positive 
ocular). 

Draw-tube by which the tube is length- 
ened or shortened. 

Main tube or body containing the draw- 
tube, and attached to the pillar by the 
arm. 

Society screw in the lower end of the 
draw-tube. 

Society screw in the lower end of the 
tube. 

Objective in position. 

Stage, under which is the substage with 
the substage condenser. 

Spring clip for holding the specimen. 



Screw for centering, and handle for the 
iris diaphragm in the achromatic con- 
denser (see Fig. 41). 

Iris diaphragm outside the principal 
focus of the condenser for use in cen- 
tering (§81). 

Mirror with plane and concave faces. 

Horse-shoe base. 

Rack and pinion for the substage con- 
denser 

Jointed pillar. 

Part of pillar with spiral spring of fine 
adjustment. 

Screw of fine adjustment. 

Milled head of coarse adjustment. 



THE MICROSCOPE 



AND 



MICROSCOPICAL METHODS 



CHAPTER I 



THE MICROSCOPE AND ITS PARTS 



APPARATUS AND MATERIAL FOR THIS CHAPTER 

A simple microscope (§ 2, 11) ; A compound microscope with nose-piece (Figs. 
70-80); eye-shade (Fig. 60), achromatic ($20), apochromatic ($22), dry (§17), 
immersion ($ 18), unadjustable and adjustable objectives ($ 23, 24) ; Huygenian or 
negative (I 38), positive ($37) and compensation oculars ($39) ; stage microme- 
ter (Ch. IV) ; homogeneous immersion liquid (§18) ; mounted letters or figures 
(£53) I ground-glass and lens paper (J 53). 

A MICROSCOPE 

§ I.- A Microscope is an optical apparatus with which one may obtain a clear 
image of a near object, the image being always larger than the object ; that is, it 
enables the eye to see an object under a greatly increased visual angle, as if the 
object were brought very close to the eye without affecting the distinctness of 
vision. Whenever the microscope is used for observation, the eye of the observer 
forms an integral part of the optical combination (Figs. 16, 21). 

\ 2. A Simple Microscope. — With this an enlarged, erect image of an object 
may be seen. It always consists of one or more converging lenses or lens-systems 
(Figs. 16-20), and the object must be placed within the principal focus (§ 11). 
The simple microscope may be held in the hand or it may be mounted in some 
way to facilitate its use (Figs 17-20). 



MICROSCOPE AND ACCESSORIES 



\CH. I 




Figs. 1-9, showing the Principal Optic Axis and the Optical Center of various 
forms of Lenses. 

Axis. The Principal Optic Axis, c-c' '. Centers of curvature of the two sur- 
faces of the lens, c. I. Optical center of the lens, r-r' '. Radii of curvature of 
the two lens surfaces, t-t' ' . Tangents in Fig. 4. 



\ 3. Principal Optic Axis. — In spherical lenses, i. e., lenses whose surfaces 
are spherical, the Axis is a line joining the centers of curvature and indefinitely 
extended. In the lens it is the unbroken part of the line c-c' in the figures. In 
lenses with one plane surface (Figs. 3, 6, 7) the radius of the plane surface is any 
line at right angles to it, but in determining the axis it must be the one which is 
continuous with the radius of the curved surface, consequently the axis in such 
lenses is on the radius of the curved surface which meets the plane surface at right 
angles. 

\ 4. Optical Center. — The optical center of a lens is the point through which 
rays pass without angular deviation, that is, the emergent ray is parallel to the 



[CH. I 



MICROSCOPE AND ACCESSORIES 



incident ray. It is determined geometrically by drawing parallel radii of the 
curved surfaces, r-r r in Figs. 4-9, and joining the peripheral ends of the radii. 
The optical center is the point on the axis cut by the line joining the peripheral 
ends of the parallel radii of the two lens surfaces. In Figs. 4-5 it is within the 
lens ; in 6-7 it is at the curved surface, and in the meniscus (8, 9) it is wholly out- 
side the lens, being situated on the side of the greater curvature. 

In determining the center in a lens with a plane surface, the conditions can 
be satisfied only by using the radius of the curved surface which is continuous 
with the axis of the lens, then an)' line at right angles to the plane surface will 
be parallel with it, and may be considered part of the radius of the plane surface. 
(That is, a plane surface may be considered part of a sphere with infinite radius, 
hence any line meeting the plane surface at right angles may be considered as the 
peripheral part of the radius.) In Figs. 6, 7, {r / ) is the radius of the curved sur- 
face and (r) of the plane surface ; and the point where a line joining the ends of 
these radii crosses the axis is at the curved surface in each case. 

By a study of Fig. 4 it will be seen that if tangents be drawn at the peripheral 
ends of the parallel radii, the tangents will also be parallel and a ray incident at 
one tangential point and traversing the lens and emerging at the other tangential 
point acts as if traversing, and is practically traversing a piece of glass which has 
parallel sides at the point of incidence and emergence, therefore the emergent ray 
will be parallel with the incident ray. This is true of all rays traversing the center 
of the lens. 

Thick Lenses. — In all of the diagrams of lenses and the course of rays through 
them in this book the lenses are treated as if they were infinitely thin. In thick 
lenses like those figured, while there would be no angular deviation for rays trav- 
ersing the center of the lens, there would be lateral displacement. This is shown 
in Fig. 57 illustrating the effect of the cover-glass. 

\ 5. Secondary Axis. — Every ray traversing the center of the lens, except the 
principal axis, is a secondary axis ; and every secondary axis is more or less 
oblique to the principal axis. In Fig. 14, line (2), is a secondary axis, and in Fig. 
15, line (1). See also Fig. 58. 



Figs. 10, 11. — Sectional views of a 
concave or diverging and a convex or 
converging lens to show that in the con- 
cave lens the principal focus is virtual as 
indicated by the dotted lines, while with 
the convex lens the focus is real and on 
the side of the lens opposite to that from 
which the light comes. 




\ 6. Principal Focus. — This is the point where rays parallel with the axis and 
traversing the lens cross the axis ; and the distance from the focus to the center of 
the lens measured along the axis is the Principal Focal Distance. In the diagrams, 
Fig. 10 is seen to be a diverging lens and the rays cross the axis only by being pro- 
jected backward. Such a focus is said to be virtual, as it has no real existence. In 



MICROSCOPE AND ACCESSORIES 



\CH. I 



Fig. 1 1 the rays do cross the axis and the focus is said to be real. If the light 
came from the opposite direction it would be seen that there is a principal focus 
on the other side, that is there are two principal foci, one on each side of the lens. 
These two foci are both principal foci, but they will be equally distant from the 
center of the lens only when the curvature of the two lens surfaces are equal. 
There may be foci on secondary axes also, each focus on a secondary axis has its 
conjugate. In the formation of images the image is the conjugate of the object 
and conversely the object is the conjugate of the image. 




Fig. 12. — Double Convex Lens, Showing Chromatic Aberration. 

The ray of white light (w) is represented as dividing into the short waved, blue s 
(b) and the long waved, red (r) light. The blue (b) ray comes to a focus nearer 
the lens and the red ray ( r) farther from the lens than the principal focus ( J) . 
Principal focus {f) for rays very near the axis ; f f and f" , foci of blue and red 
light coming from near the edge of the lens. The intermediate wave lengths 
7vould have foci all the way between f and f" '. 

| 7. Chromatic Aberration — This is due to the fact that ordinary light con- 
sists of waves of varying length, and as the effect of a lens is to change the direc- 
tion of the waves, it changes the direction of the short waves more markedly 
than the long waves. Therefore, the short waved, blue light will cross the axis 
sooner than the long waved, red light, and there will result a superposition of 
colored images, none of which are perfectly distinct (Fig. 12). 

Fig. 13. The ray (o) near the 
edge of the lens is brought to a 

focus nearer the lens than the 
ray (i). Both are brought to 
a focus sootier than rays very 
near the axis, (f) Principal 

focus for rays very near the 
axis; (f) Focus for the ray 
(i), and {f /,s ) Focus for the ray 
(o). Intermediate rays would 
cross the axis all the way from 
(f'tof). 




Fig. 13. 



Double Convex Lens, showing 
Spherical Aberration. 



\ 8. Spherical Aberration.— This is due to the unequal turning of the light 
in different zones of a lens. The edge of the lens refracts proportionally too 
much and hence the light will cross the axis or come to a focus nearer the lens 
than a ray which is nearer the middle of the lens. Thus, in Fig. 13, if the focus 



CH. /] 



MICROSCOPE AND ACCESSORIES 



of parallel rays very near the axis is at/", rays (o i), nearer the edge, would come to 
a focus nearer the lens, the focus of the ray nearest the edge being nearest the lens. 

\ 9. Correction of Chromatic and of Spherical Aberration. — Every simple 
lens has the defect of both chromatic and spherical aberration, and to overcome 
this, kinds of glass of different refractive power and different dispersive power 
are combined, concave lenses neutralizing the defects of convex lenses. If the 
concave lens is not sufficiently strong to neutralize the aberrations of the convex 
lens, the combination is said to be under-corrected, while if it is too strong and 
brings the marginal rays or the blue rays to a focus beyond the true principal 
focus, the combination is over-corrected. 

In Newton's time there was supposed to be a direct proportion between the 
refractive power of any transparent medium and its dispersive power ( i. e. its power 
to separate the light into colors). If this were true then the contention of Newton 
that it would be impossible to do away with the color without at the same time 
doing away with the refraction would be true and useful achromatic combinations 
would be impossible. It was found by experiment, however, that there is not a 
direct ratio between the refractive and dispersive powers for the different colors 
in different forms of glass, so that it is possible to do away largety with chromatic 
aberration and retain sufficient refraction to make the combination serve for the 
production of images. ( See also the discussion under apochromatic objectives \ 22 ) 

Probably no higher technical skill is used in any art than is requisite in the 
preparation of microscopical objectives, oculars and illuminators. 

Figs. 14 and 15. 14. Convex lens 
showing the position of the object ( A-B ) 
outside the principal focus (E), and 
the course of the rays in the formation 
of real images. To avoid confusion the 
rays are drawn from only one point. 

A B. Object outside the principal 
focus. B' A' . Real, enlarged image 
on the opposite side of the lens. 

Axis. Principal optic axis. 1,2,3. 
Rays after traversing the lens. They 
are converging , and consequently form 
a real image. The dotted line and the 
line ( 2 ) give the direction of the rays as 
if unaffected -by the lens. (E). The 
principal focus . 

Fig. 15. — Convex lens, showing the 
position of the object {A B) within the 
principal focus and the course of rays 
in the formation of a virtual linage. 




14 



15 



A B. The object placed between the lens and its focus; A' B' virtual image 
formed by tracing the rays backward. It appears on the same side of the lens as 
the object, and is erect ( \ 11). 

Axis. The principal optic axis of the lens. F. The principal focus. 

1 ', 2, 3. Rays from the point B of the object. They are diverging after trav- 
ersing the lens, but not so diveigent as if no lens were present, as is shown by the 



MICROSCOPE AND ACCESSORIES 



[CH. I 



dotted tines. Ray (/) traverses the center of the lens, and is therefore not deflected. 
It is a secondary axis {\ 5) . 

\ 10. Geometrical Construction of Images. — As shown in Figs. 14-15, for the 
determination of any point of an image, or the image being known, to determine 
the corresponding part of the object, it is necessary to know the position of the 
principal focus (and there is one on each side of the lens, § 6), and the optical 
center (Figs. 1-9 of the lens). Then a secondary axis (2) in Fig. 14, (1) in Fig. 
15, is drawn from the extremity of the object and prolonged indefinitely above the 
lens, or below it for virtual images. A second line is drawn from the extremity of 
the object, (3) in Fig. 14, (2) in Fig. 15, to the lens parallel with the principal 
axis. After traversing the lens it must be drawn through the principal focal point. 
If now it is prolonged it will cross the secondary axis above the lens for a real 
image and below for a virtual image. The crossing point of these lines determines 
the position of the corresponding part of the image. Commencing with any point 
of the object the corresponding point of the image may be determined as just 
described, and conversely commencing with the image, corresponding points of 
the object may be determined. 



SIMPLE MICROSCOPE : EXPERIMENTS 



§H 



Employ a tripod or other simple microscope, and for object 
a printed page. Hold the eye about two centimeters from the upper 
surface of the magnifier, then alternately raise and lower the magnifier 
until a clear image may be seen. (This mutual arrangement of micro- 
scope and object so that a clear image is seen, is called focusing). 
When a clear image is seen, note that the letters appear as with the 
unaided eye except that they are larger, and the letters appear erect or 
right side up, instead of being inverted, as with the compound 
microscope (§ 12). 

Fig. 16. Diagram of the simple microscope show- 
ing the course of the rays and all the images, and 
that the eye forms an integral part of it. 

A 1 B l . The object within the principal focus. A 3 
B 3 . The virtual image on the same side of the lens 
as the object. It is indicated with dotted lines, as it 
has no actual existence. 

B- A-. Retinal image of the object (A 1 B 1 ). The 
virtual image is simply a projection of the retinal 
image in the field of vision. 

Axis. The principal optic axis of the micro- 
scope and of the eye. Cr. Cornea of the eye. L. 
Crystalline lens of the eye. R. Ideal refracting 
surface at which all the refractions of the eye may 
be assumed to take place. 




CH. /] 



MICROSCOPE AND ACCESSORIES 



Hold the simple microscope directly toward the sun and move it 
away from and toward a piece of printed paper until the smallest 
bright point on the paper is obtained. This is the burning point 
or focus, and as the rays of the sun are nearly parallel, the burning 
point represents approximately the principal focus (Fig. n). With- 
out changing the position of the paper or the magnifier, look into the 
magnifier and note that the letters are very in- 
distinct or invisible. Move the magnifier a 
centimeter or two farther from the paper and no 
image can be seen. Now move the magnifier 
closer to the paper, that is, so that it is less than 
the focal distance from the paper, and the letters 
will appear distinct. This shows that in order 
to see a distinct image with a simple microscope, 
the object must always be nearer to it than its 
principal focal point. Or, in other words, the 
object must be within the principal focus. Com- 
pare (§53). 

After getting as clear an image as possible with a simple micro- 
scope, do not change the position of the microscope but move the eye 
nearer and farther from it, and note that when the eye is in one posi- 
tion, the largest field may be seen. This position corresponds to the 
eye-point (Fig. 30) of an ocular, and is the point at which the largest 
number of rays from the microscope enter the eye. Note that the 
image appears on the same side of the magnifier as the object. 




Fig. 17. 

Tripod Magnifier. 




Fig. 19. Lens-holder ( The Bausch & 
Lomb Optical Co. ) 



Fig. 18. The Hastings Aplan- 
atic Triplet. ( The Bausch & Lomb 
Optical Co. ) 



MICROSCOPE AND ACCESSORIES 



\CH. I 




Fig. 20. Dissecting Microscope. This is simply a device for holding the lens 
and the object to be observed. ( The Bansch & Lomb Optical Co.) 

. Simple microscopes are very convenient when only a small mag- 
nification (Ch. IV) is desired, as for dissecting. Achromatic triplets 
are excellent and convenient for the pocket. For use in conjunction 
with a compound microscope, the tripod magnifier (Fig. 17) is one of 
the best forms. For man5' purposes a special mechanical mounting is 
to be preferred. 

COMPOUND MICROSCOPE 

\ 12. A Compound Microscope. — This enables one to see an enlarged, in- 
verted image. It always consists of two optical parts — an objective, to produce an 
enlarged, inverted, real image of the object, and an ocular acting in general like 
a simple microscope to magnify this real image (Fig. 21 ). There is also usually 
present a mirror, or both a mirror and some form of condenser or illuminator for 
lighting the object. The stand of the microscope consists of certain mechanical 
arrangements for holding the optical parts and for the more satisfactory use of 
them. (See frontispiece. ) 

\ 13. The Mechanical Parts of a laboratory, compound microscope are shown 
in'the frontispiece, and are described in the explanation of that figure. The stu- 



CH. /] 



MICROSCOPE AND ACCESSORIES 



dent should stud} - the figure with a microscope before him and become thoroughly 
familiar with the names of all the parts. See also the cuts of microscopes at the 
end of Ch. II. 



OPTICAL PARTS 

\ 14. Microscopic Objective. — This 
consists of a converging lens or of one 
or more converging lens-systems, which 
give an enlarged, inverted, real image of 
the object ( Figs. 14, 21 ). And as for the 
formation of real images in all cases, 
the object must be placed outside the 
principal focus, instead of within it, as 
for the simple microscope. (See \\ n, 
53, Figs. 16, 2i.) 

Modern microscopic objectives usu- 
ally consist of two or more systems or 
combinations of lenses, the one next 
the object being called the front com- 
bi?iation or lens, the one farthest from 
the object and nearest the ocular, the 
back combination or system. There may 
be also one or more intermediate sys- 
tems. Each combination is, in general, 
composed of a convex and a concave 
lens. The combined action of the sys- 
tem serves to produce an image free 
from color and from spherical distor- 
tion. In the ordinary achromatic ob- 
jectives the convex lenses are of crown 
and the concave lenses of flint glass 
(Figs. 22, 23). 

Fig. 21. Diagram showing the 
principle of a compound microscope with 
the course of the rays from the object 
(A B) through the objective to the real 
image ( B' A'), thence through the ocu- 
lar a?id into the eye to the retinal image 
{A-B 2 ), and the projection of the retinal 
image into the field of vision as the 
virtual image {B 3 Ai). 

A B. The object. A 2 B 2 . The retinal 
image of the inverted real image, {B l A z ) , 
formed by the objective. B^A?. The 
inverted virtual image, a projection of 
the retinal image. 



B J -^».-± 




i- :$a* 



IO 



MICROSCOPE AND ACCESSORIES 



\_CH.I 



Axis. The principal optic axis of the microscope and of the eye. 

Cr. Cornea of the eye. L. Crystalline lens of the eye. R. Single, ideal, re- 
fracting surface at which all the refractions of the eye may be assumed to take 
place. 

F. F. The principal focus of the positive ocular and of the objective. 

Mirror. The mirror reflecting parallel rays to the object. The light is central. 
See Ch. II. 

Pos. Ocular. An ocular in which the real image is formed outside the ocular. 
Compare the positive ocular with the simple microscope {Fig. 16). 

NOMENCLATURE OR TERMINOLOGY OF OBJECTIVES 

\ 15. Equivalent Focus. — In America, England, and sometimes also on the 
Continent, objectives are designated by their equivalent focal length. This length 
is given either in inches (usually contracted to in. ) or in millimeters (mm.) Thus: 
An objective designated jV in. or 2 mm., indicates that the objective produces a 
real image of the same size as is produced by a simple converging lens whose 
principal focal distance is ^ inch or 2 millimeters (Fig. 11). An objective 
marked 3 in. or 75 mm., produces approximately the same sized real image as a 
simple converging lens of 3 inches or 75 millimeters focal length. And in accord- 
ance with the law that the relative size of object and image vary directly as their 
distance from the center of the lens (Figs. 14, 15, see Ch. IV,) it follows that the 
less the focal distance of the simple lens or of the equivalent focal distance of the 
objective, the greater is the size of the real image, as the tube-length remains con- 
stant and the image in all cases is found at about 160 or 250 mm. from the objective. 
\ 16. Numbering or Lettering Objectives. — Instead of designating objectives 
by their equivalent focus, many Continental opticians use letters or figures for this 
purpose. With this method the smaller the number, or the earlier in the alpha- 
bet the letter, the lower is the power of the objective. (See further in Ch. IV, for 
the power or magnification of objectives). This method is entirely arbitrary and 
does not, like the one above, give direct information concerning the objective. 

I 17. Air or Dry Objectives. — These are objectives in which the space be- 
tween the front of the objective and the object or cover-glass is filled with air 
(Fig. 22). Most objectives of low and medium power (z. e,, \ in. or 3 mm. and 
lower powers) are dry. 

Fig. 22. Section of a dry objective showing 
working distance and lighting by reflected 
light. 

Axis. The principal optic axis of the ob- 
jective. 

B C. Back Combination, composed of a 
plano-concave lens of flint glass {F), and a 
double convex lens of crown glass (c). 
F C. Front Combination. 
C, O, si. The cover-glass, object and slide. 
Mirror. The mirror is represented as above 
the stage, and as reflecting parallel rays from 
its plane face upon the object. 

Stage. Section of the stage of the microscope. 




CH. /] 



MICROSCOPE AND ACCESSORIES 



II 



W. The Working Distance, that is the distance from the front of the objective 
to the object when the objective is in focus. 

\ iS. Immersion Objectives. — An immersion objective is one with which 
there is some liquid placed between the front of the objective and the object or 
cover-glass. The most common immersion objectives are those (A) in which 
water is used as the immersion fluid, and (B) where some liquid is used having the 
same refractive and dispersive power as the front lens of the objective. Such a 
liquid is called homogeneous, as it is optically homogeneous with the front glass of 
the objective. It may consist of thickened cedar wood oil or of glycerin contain- 
ing some salt, as stannous chlorid in solution. When oil is used as the immersion 
fluid the objectives are frequently called oil immersion objectives. The disturb- 
ing effect of the cover-glass (Fig. 57) is aimost wholly eliminated by the use of 
homogeneous immersion objectives, as the rays undergo very little or no refraction 
on passing from the cover-glass through the immersion medium and into the ob- 
jective ; and when the object is mounted in balsam there is practically no refrac- 
tion in the ray from the time it leaves the balsam till it enters the objective. 

Fig. 23. Sectional view of an Immersion, Ad- 
justable Objective, and the object lighted with axial 
or central and with oblique light. 

Axis. The principal optic axis of the objective. 

B C, M C, F C. The back, middle and front 
combination of the objective. In this case the 
front is not a combination, but a single plano- 
convex lefts. 

A, B. Parallel rays reflected by the mirror 
axially or centrally upon the object. 

C Ray reflected to the object obliquely. 

I. Immersion fluid between the front of the 
objective and the cover glass or object (O). 

Mirror. The mirror of the microscope . 

O. Object. It is represented without a cover- 
glass. Ordinarily objects are covered whether ex- 
amined with immersion or with dry objectives. 

Stage. Section of the stage of the fnicroscope. 

\ 19. Non-Achromatic Objectives.— These are objectives in which the chro- 
matic aberration is not corrected, and the image produced is bordered by colored 
fringes. They show also spherical aberration and are used only on very cheap 
microscopes. {\\ 7, 8, Figs. 12, 13). 

I 20. Achromatic Objectives.— In these the chromatic and the spherical aber- 
ration are both largely eliminated by combining concave and convex lenses of dif- 
ferent kinds of glass "so disposed that their opposite aberrations shall correct 
each other." All the better forms of objectives are achromatic and also aplanatic. 
That is the various spectral colors come to the same focus. 

jj 21. Aplanatic Objectives, etc.— These are objectives or other pieces of 
optical apparatus (oculars, illuminators, etc. ), in which the spherical distortion is 




12 MICROSCOPE AND ACCESSORIES \_CH. I 

wholly or nearly eliminated, and the curvatures are so made that the central and 
marginal parts of the objective focus rays at the same point or level. Such pieces 
of apparatus are usually achromatic also. 

| 22. Apochromatic Objectives. — A term used by Abbe to designate a form of 
objective made by combining new kinds of glass with a natural mineral (Calcium 
fluorid, Fluorite, or Fluor spar). The name, Apochromatic, is used to indicate 
the higher kind of achromatism in which rays of three spectral colors are com- 
bined at one focus, instead of rays of two colors as in the ordinary achromatic ob- 
jectives. At the present time (1901) several opticians make apochromatic ob- 
jectives without using the fluorite. Some of the early apochromatics deteriorated 
rather quickly in hot moist climates. Those now made are quite permanent. 

The special characteristics of these objectives, when used with the "compen- 
sating oculars" are as follows : 

(1) Three rays of different color are brought to one focus, leaving a small ter- 
tiary spectrum only, while with objectives as formerly made from crown and flint 
glass, only two different colors could be brought to the same focus. 

(2) In these objectives the correction of the spherical aberration is obtained 
for two different colors in the brightest part of the spectrum, and the objective 
shows the same degree of chromatic correction for the marginal as for the central 
part of the aperture. In the old objectives, correction of the spherical aberration 
was confined to rays of one color, the correction being made for the central part of 
the spectrum, the objective remaining zwzfl^r-corrected spherically for the red rays 
and C27<?r-corrected for the blue rays (§9). 

(3) The optical and chemical foci are identical, and the image formed by 
the chemical rays is much more perfect than with the old objectives, hence the 
new objectives are well adapted to photograph}'. 

(4) These objectives admit of the use of very high oculars, and seem to be a 
considerable improvement over those made in the old way with crown and flint 
glass. According to Dippel (Z. w. M. 1886, p. 300) dry apochromatic objectives 
give as clear images as the same power water immersion objectives of the old form. 

\ 23. Non-Adjustable or Unadjustable Objectives. — Objectives in which the 
lenses or lens systems are permanently fixed in their mounting so that their rela- 
tive position always remains the same. Low power objectives and those with 
homogenous immersion are mostly non-adjustable. For beginners and those un- 
skilled in manipulating adjustable objectives ($ 24), non- adjustable ones are more 
satisfactory, as the optician has put the lenses in such a position that the most 
satisfactory results may be obtained when the proper thickness of cover-glass and 
tube-length are emplo^'ed. (See table of tube-length and thickness of cover-glass 
below, p. 14.) 

\ 24. Adjustable Objectives. — An adjustable objective is one in which the dis- 
tance between the systems of lenses (usually the front and the back systems) may 
be changed by the observer at pleasure. The object of this adjustment is to cor- 
rect or compensate for the displacement of the rays of light produced by the 
mounting medium and the cover-glass after the rays have left the object. It is 
also to compensate for variations in "tube-length." See $29. As the displace- 
ment of the rays by the cover-glass is the most constant and important, these ob- 
jectives are usually designated as having cover-glass adjustment or correction. 
(Fig. 23. See also practical work with adjustable objectives, Ch. II). 



CH. 7] MICROSCOPE AND ACCESSORIES 13 

I 25. Parachromatic, Pantachromatic and Semi-apochromatic Objectives. — 
These are trade names for objectives, most of them containing one or more lenses 
of the new glass (§ 22). They are said to approximate much more closely to the 
apochromatics than to the ordinary objectives. 

\ 26. Variable Objective. — This is a low power objective of 36 to 26 mm. 
equivalent focus, depending upon the position of the combinations. By means of a 
screw collar the combinations may be separated, diminishing the power, or ap- 
proximated and thereby increasing it. 

\ 27. Projection Objectives. — These are designed especially for projecting an 
image on a screen and for photo-micrography. They are characterized by having 
a fiat, sharp field brilliantly lighted. (See Ch. IV, IX. ) 

\ 28. Illuminating or Vertical Illuminating Objectives. — These are designed 
for the study of opaque objects with good reflecting surfaces, like the rulings on 
metal bars and broken or polished and etched surfaces of metals employed in 
micro-metallography. The light enters the side of the tube or objective and is 
reflected vertically downward through the objective and thereby is concentrated 
upon the object. The object reflects part of the light back into the microscope 
thus enabling one to see a clear image. 

\ 29. Tube-Length and Thickness of Cover-Glasses. — "In the construction 
of microscopic objectives, the corrections must be made for the formation of the 
image at a definite distance, or in other words the tube of the microscope on 
which the objective is to be used must have a definite length. Consequently the 
microscopist must know and use this distance or 'microscopical tube-length' to 
obtain the best results in using any objective in practical work." Unfortunately 
different opticians have selected different tube-lengths and also different points 
between which the distance is measured, so that one must know what is meant by 
the tube-length of each optician whose objectives are used. See table. 

The thickness of cover-glass used on an object (See Ch. VII, on mounting), 
except with homogeneous immersion objectives, has a marked effect on the light 
passing from the object (Fig. 57). To compensate for this the position of the sys- 
tems composing the objective are closer together than they would be if the object 
were uncovered. Consequently, in non-adjustable objectives some standard thick- 
ness of cover-glass is chosen by each optician and the position of the systems 
arranged accordingly. With such an objective the image of an uncovered object 
would be less distinct than a covered one, and the same result would follow the 
use of a cover-glass much too thick. 



H 



MICROSCOPE AND ACCESSORIES 



\CH. I 



Length in Millimeters and Parts included in "Tube-Length" by 
Various Opticians* 
Pts. included 
in "Tube- 'Tube-Length" in 

length." Millimeters. 

See Diagram. 

f E. Leitz, Wetzlar 170 mm. 

I Natchet et Fils, Paris 160 mm. 

j Powell and Lealand, London 254 mm. 

j C. Reichert, Vienna 160 to 180 mm. 

j Spencer Lens Co., Buffalo 160 mm. 

[W. Wales, New York 254 mm. 

f Bausch & Lomb Opt. Co., Rochester 160 or 216mm. 

I Bezu, Hausser et Cie, Paris 180 mm. 

I Klonne und Miiller, Berlin 160-180 or 254mm. 

-j W. & H. Seibert, Wetzlar 170 mm. 

j Swift & Son, London 165 to 228^ mm. 

I C. Zeiss, Jena 160 or 250 mm. 

[R. Winkel, Gottingen 192 mm. 

. Gundlach Optical Co., Rochester 254 mm. 

. Ross & Co., London 254 or 160 mm. 

. Queen & Co., Philadelphia 160 mm. 

. R. & J. Beck, London 254 or 160 mm. 

. Hartnack, Potsdam, Germany i6oandi8omm. 

Verick (Stiassnie) Paris 160-200 mm. 

Watson & Sons, London 160-250 mm. 

J. Zentmayer, Philadelphia 160-235 mm. 

Thickness of Cover-Glass for Which Non-Adjustable Objectives are Corrected by 

Various Opticians. 
j Powell and Lealand, London, 
roo mm. \ w. Wales, New York. 

r 2 5 °g- mm. Watson & Sons, London. 

T - J E. Leitz, Wetzlar. 

1 o'o mm. j r, Winkel, Gottingen, Germany. 

'-XTrf- mm. Ross & Co., London. 

f Klonne und Miiller, Berlin. 
18 j Spencer Lens Co., Buffalo. 

roo mm. 1 Bausch & Lomb Optical Co., Rochester. 

[Queen & Co., Philadelphia, 
mm. C. Zeiss, Jena, 
mm. C. Reichert, Vienna. 

(Gundlach Optical Co., Rochester. 
W. and H. Seibert, Wetzlar. 
R. and J. Beck, London, 
mm. J. Zentmayer, Philadelphia. 
j Nachet et Fils, Paris. 
\ Bezu, Hausser et Cie, Paris. 
Swift and Son, London. 
E. Hartnack, Potsdam, German)'. 




"10" (i 

-5.-1 i 

"1 IT 



l_0J_'l 5. mm 

r V o mm. 
-? oh- mm. 



*The information contained in these tables was very kindty furnished by the 
opticians named, or obtained by consulting catalogs. In most of the later catalogs 
the information is definite, and many makers now not only put their names and the 
equivalent focal length on their objectives, but they add the numerical aperture 
(3 31) and the tube-length for which the objective is corrected. This is in accord- 
ance with the recommendations of the author in the original paper on "tube- 
length," (Proc. Amer. Soc. Micr., Vol. IX., p. 168, also by Bausch, Vol. XII, p. 
43). If the table in this edition is compared with the original table or with that in 
the previous edition of this book some differences will be noted, the changes being 



Owing to information received after the table on p. 14 was printed it is neces- 
sary to replace that table bv one containing the latest information. In this revised 
table tube-length b-d of the diagram greatly preponderates, and the great majority 
of unadjustable objectives are corrected for a thickness of cover-glass falling be- 
tween fifteen and twenty one hundredths of a millimeter (0.15-0.20 mm.). 

Length in Millimeters and Parts included tn the "Tube-Length" by 
, Various Opticians. 

Pts. included 
in '"Tube- 
length." 

See Diagram. 



"Tube-Length" in 
Millimeters. 




[Chas. Baker, London, England ._. 

The Bausch & Lomb Optical Co., 

Rochester, N. Y. . _ 

R. & J. Beck, London, England .. 

Bezu, Hausser & Cie, Paris, France 

Klonne und Miiller, Berlin, Germany. _. 
Queen & Co., Incorporated, Phila., Pa. _ 

Ross, Ltd , London, England 

W. und H. Seibert, Wetzlar, Germany.. 

Swift & Son, London, England 

Watson & Sons, London, England 

R. Winkel, Goettingen, Germany 

Carl Zeiss, Jena, Germany 



a-d 



f Ernst Leitz, Wetzlar, Germany 

J Nachet et Fils, Paris, France 

j Powell & Lealand, London, England 

j C. Reichert, Vienna, Austria 

I Spencer Lens Company, Buffalo, N. Y 

I W. Wales, New York' 



The Gundlach Opt. Co., Rochester, N. Y. 
E. Hartnack, Potsdam, Germany.. 



Dollond & Co., London, England 
Verick (Stiassnie) Paris, France. _._ 
P. Waechter, Berlin-Friedenau, Germany 
J. Zentmayer, Philadelphia, Pa. 



150 or 250 mm. 

160 or 216 mm. 
.160 or 220 mm. 

180 mm. 

160 or 250 mm. 
b-d [ Queen & Co., Incorporated, Phila., Pa 170 mm. 

160 or 254 mm. 
.170 mm. 

160 or 228 mm. 

160 or 250 mm. 
. 192 mm. 
.160 or 250 mm. 

170 mm. 

160 mm. 

254 mm. 

1 60- 1 80 mm . 
.160 mm. 
.254 mm. 

254 mm. 

160 mm. 

165, 240 mm. 
.160-200 mm. 
.160 mm. 

160 or 235 mm. 

Thickness of Cover-Glass for Which Non- Adjustable Objectives are Corrected by 

Various Opticians 

f The Bausch & Lomb Optical Co., Rochester, N. Y. 

] Klonne und Miiller, Berlin, Germany. 

I Queen & Co., Incorporated, Philadelphia, Pa. 

I The Spencer Lens Co., Buffalo, N. Y. 

( Ernst Leitz, Wetzlar, Germany. 

-] P. Wachter, Berlin-Friedenau, Germany. 

( R. Winkel, Goettingen, Germany. 

f Chas. Baker, London, England. 

] R. .&. J. Beck, Ltd., London, England. 

J Gundlach Optical Co. , Rochester, N. Y. 

I W. und H. Seibert, Wetzlar, Germany. 

J E. Hartnack, Potsdam, Germany. 

t C. Reichert, Vienna, Austria. 

(Ross, Ltd., London, England. 

X Verick (Stiassnie), Paris, France. 

I Carl Zeiss, Jena, Germany. 
J. Zentmayer, Philadelphia, Pa. 

I Dollond & Co., London, England. 

1 Nachet et Fils, Paris, France. 
Bezu Hausser & Cie, Paris, France. 

j Powell & Lealand, London, England. 

I. Swift & Son, London, England. 
0.20 mm. Watson & Sons, London, England. 

0.25 mm. W. Wales, New York. 



0.18 mm. 



0.17 mm. 



o. 15 mm. 

0.15-0. 18 mm. 

0.15-0.20 mm. 

o. 12-0. 17 mm. 
0.10-0. 15 mm. 
o. 10-0. 12 mm. 
o. 10 mm. 



CH. /] 



MICROSCOPE AND ACCESSORIES 



15 



m 



§ 30. Aperture of Objectives. — The angular aperture or angle 
of aperture of an objective is the "angle contained, in each case, be- 
tween the most diverging of the rays issuing from the axial point of an 
object [/. <?. , a point in the object situated on the optic axis of the 
microscope], that can enter the objective and take part in the formation 
of an image." (Carpenter). 

in the direction of uniformity and in general in the direction recommended by the 
writer and Mr. Bausch and the committee of the American Microscopical Society. 
The recommendations of the committee, published in the Proceedings, Vol. XII., 
p. 250, are as follows : 

"Believing in the desirability of a uniform tube-length for microscopes, we 
unanimously recommend : 1. That the parts of the microscope included in the 
tube-length should be the same by all opticians, and that the parts included should 
be those between the upper end of the tube where the ocular is inserted and the 
lower end- of the tube where the objective is inserted. 

2. That the actual extent of tube 
length as defined in section 1 — Be, for the 
short or continental tube, 160 mm., or 6.3 
inches, and 216 mm., or S}4 inches, for 
the long tube, and that the draw tube of 
the microscope possess two special 
marks indicating these standard lengths. 

3. That oculars be made par-focal, 
and that the par-focal plane be coincident 
with that of the upper end of the tube. 

4. That the mounting of all object- 
ives of 6 mm. ( % inch) and shorter focus 
should be such as to bring the optical 
center of the objective 1% inches below 
the shoulder, and that all objectives be 
marked with the tube-length for which 
they are corrected. 

5. That non-adjustable objectives be 
corrected for cover-glass from $fc to t 2 °q 
mm. { T \jj to T fp inch) in thickness. 

These recommendations give a dis- 
tance of 10 inches (254 mm.) between the 
par-focal plane of the ocular and the op- 
tical center of the objective for the long 
tube, and are essentially in accord with 
the actual practice of opticians. 

At the request of the committee, a 
joint conference was held with the opti- 



SHI 




Fig. 25. The tube of a microscope with 
ocular micrometer and nose piece in 
position to show that in measuring 
tube-length one must measure from 
the eye lens to the place where the ob- 
jective is attached. {Zeiss' Catalog.) 

cians belonging to the Society and present at the meeting. They expressed their 
belief in the entire practicability of the above recommendations and a willingness 
to adopt them. ' ' 

(Signed) Simon H. Gage, 

A. Clifford Mercer, 
Charles E. Barr. 




1 6 MICROSCOPE AND ACCESSORIES \CH. I 

In general the angle increases with the size of the lenses forming the objective 
and the shortness of the equivalent focal distance {\ 15). If all objectives were 
dry or all water or all homogeneous immersion a comparison of the angular aper- 
ture would give one a good idea of the relative number of image forming rays 

Fig. 26. Diagram illustrating the angular aperture of 
a' microscopic objective. Only the front lens of the objective 
is shown. 

Axis. The principal optic axis of the objective. 

B A, B C, the most divergent rays that can enter the 
objective, they mark the angular aperture. A B D or C B 
D half the angular aperture. This is designated by u in 
making Numerical Aperture computations. See the table, \ 33. 

transmitted by different objectives ; but as some are dry, 
others water and still others homogeneous immersion, one 
can see at a glance that, other things being equal, the dry 
objective (Fig. 27) receives less light than the water immersion, and the water im- 
mersion (Fig. 28) less than the homogeneous immersion (Fig. 29). In order to 
render comparison accurate between different kinds of objectives, Professor 
Abbe takes into consideration the rays actually passing from the back combi- 
nation of the objectives to form the real image ; he thus takes into account the 
medium in front of the objective as well as the angular aperture. The term 
'Numerical Aperture,' 1 '' (N A.) was introduced by Abbe to indicate the capacity 
of an optical instrument "for receiving rays from the object and transmitting 
them to the image. 

\ 31. Numerical Aperture (abbreviated N. A.), as now employed for micro- 
scope objectives, is the ratio of the semi-diameter of the emergent pencil to the 
focal length of the lens. Or as the factors are more readily obtainable it is sim- 
pler to utilize the relationship shown in the La Grange-Helmholtz-Abbe formula, 
and indicate the aperture by the expression : N. A.=n sin u. In this formula ?i 
is the index of refraction of the medium in front of the objective (air, water or 
homogeneous liquid), and sin u is the sine of half the angle of aperture ( Fig. 26, 
DBA). For the mathematical discussion showing that the expressions 

semi-diameter of emergent pencil . , , x . , , _ 

— = — 5 , — ; — ; — = n sm u, the student is referred to the lournal 

focal length of the lens 

of the Royal Microscopical Society, 1881, pp. 392-395, 1898, p. 363. 

For example, take three objectives each of 3 mm. equivalent focus, one being 
a dry, one a water immersion, and one a homogeneous immersion. Suppose that 
the dry objective has an angular aperture of 106 , the water immersion of 94 and 
the homogeneous immersion of 90 . Simply compared as to their angular aper- 
ture, without regard to the medium in front of the objective, it would look as if 
the dry objective would actually take in and transmit a wider pencil of light than 
either of the others. However, if the medium in front of the objective is con- 
sidered, that is to say, if the numerical instead of the angular apertures are 
compared, the results would be as follows : Numerical Aperture of 'a dry objective 
of 106 , N. A.=?z sin u. In the case of dry objectives the medium in front of the 
objective being air, the index of refraction is unity, whence n=l. Half the 
angular aperture is -^-P- °=53°. By consulting a table of natural sines it will be 
found that the sine of 53 is 0.799, whence N. A.=» or 1 X si n u or 0.799=0.799.* 

*| 32. Interpolation. — In practice, as in solving problems similar to those 
on the following pages and those in refraction if one cannot find a sine exactly 



CH. /.] 



MICROSCOPE AND ACCESSORIES 



17 




^ 


_o^>\ 




■I //sC^ 


<A\U 


\ 1 1 // c cnrcv 


"^^4^ 


J^SZ^- 



Figs. 27-29 are somewhat modified from 
Ellenberger, and are introduced to illustrate 
the relative amount of utilized light, with dry, 
water immersion and homogeneous immer- 

27 sion objectives of the same eqtiivalent focus. 
The point from which the rays emanate is in 
air in each case. If Canada balsam were be- 
neath the cover-glass in place of the air there 
would be practically no refraction of the rays 
on entering the cover glass ( \ 18) . 

Fig. 27. Showing the course of the rays 

28 passing through a cover glass from an axial 
point of the object, and the number that 
finally enter the front of a dry objective. 

Fig. 28. Rays from the axial point of 
the object traversing a cover of the same thick- 
ness as in Fig. 27, and entering the front 
lens of a water immersion objective. 
Fig. 29. Rays from an axial point of the 

29 object traversing a cover glass and entering 
the front of a homogeneous immersion 
objective. 

With the water immersion objective the medium in front is 
water, and its index of refraction is 1.33, whence n = 1.33. Half 
the angular aperture is -^ °= 
found to be 0.731, i. e., sin u- 
or 0.731=0.972. 




: 47°, and by the table the sine of 47 is 
=0.731, whence N. A.=?z or 1.33 x sin u 



corresponding to a given angle ; or if one has an angle which does not correspond 
to any sine or angle given in the table, the sine or angle may be closely approxi- 
mated by the method of interpolation, as follows : Find the sine in the table nearest 
the sine whose angle is to be determined. Get the difference of the sines of the 
angles greater and less than the sine whose angle is to be determined. That will give 
the increase of sine for that region of the arc for 15 minutes. Divide this increase 
by 15 and it will give with approximate accuracy the increase for 1 minute. Now 
get the difference between the sine whose angle is to be determined and the sine 
just below it in value. Divide this difference by the amount found necessary for 
an increase in angle of 1 minute and the quotient will give the number of minutes 
the sine is greater than the next lower sine whose angle is known. Add this num- 
ber of minutes to the angle of the next lower sine and the sum will represent the 
desired angle of the sine. Or if the sine whose angle is to be found is nearer in 
size to the sine just greater, proceed exactly as before, getting the difference in the 
sines, but subtract the number of minutes of difference and the result will give the 
angle sought. For example take the case in Section 97 where the sine of the 
angle of 28 54/ is given as 0.48327. If one consults the table the nearest sines 
found are 0.48099, the sine of 28 45', and 0.48481, the sine of 29 . Evidently 



1 8 MICROSCOPE AND ACCESSORIES [CH. I 

I With the oil immersion in the same way N. A.= n sin u ; n or the 
index of refraction of the homogeneous fluid in front of the objective 
is 1.52, and the semi-angle of aperture is - 9 2-°-°=45°. The sine of 45 
is 0.707, whence N. A.=«. or i52Xsinzc or 0.707=1.074. 

By comparing these numerical apertures : Dry 0.799, water 0.972, 
homogeneous immersion 1.074, the same idea of the real light efficiency 
and image power of the different objectives is obtained, as in the graphic 
representations shown in Figs. 27-29. 

If one knows the numerical aperture (N. A.) of an objective the 
angular aperture is readily determined from the formula ; and one 
can determine the equivalent angles of objectives used in different 
media (z. e., dry or immersion). For example, suppose each of three 
objectives has a numerical aperture (N. A.) of 0.80, what is the an- 
gular aperture of each ? Using the formula of N. A.=?z sin ?i, one has 
N. A.= 0.80 for all the objectives. 
For the dry objective n = 1 (Refractive index of air). 

" water immersion objective tz= 1.33. (Refractive index of water), 
homogeneous immersion objective 71=1. 52 (Refractive index 
of homogeneous liquid). And 2 ti is to be found in each case. 

For the dry objective, substituting the known values the formula 
becomes 0.80= 1 sin u, or sin u = 0.80. By inspecting the table of 
natural sines (3d page of cover) it will be found that 0.80 is the sine 
of 53 degrees and 8 minutes. As this is half the angle the entire 
angular aperture of the dry objective must be 53 8'x 2 = 106 16'. 

For the water immersion objective, substituting the known values 

in the formula as before : 0.80 = 1.33 sin u, or sin u = — — = 0.6015. 

i-33 
Consulting the table of sines as before, it will be found that 0.6015 is 
the sine of 36 59' whence the angular aperture (water angle) is 36 
59'X2 = 73° 58'. 

For the homogeneous immersion objective, substituting the known 
values, the formula becomes: 0.80 = 1.52 sin u whence sin u = 

— — = 0. S26v And by consulting the table of sines it will be found 
1.52 ° ° 



then the angle sought must lie between 28 45', and 29 . If the difference between 
0.48481 and 0.48099 be obtained, 0.48481 — 0.48099 = 0.00382, andif this increase for 
I5 / be divided by 15 it will give the increase for 1 minute ; 0.00382 -5- 15 = 0.000254. 
Now the difference between the sine whose angle is to be found and the next 
lower sine is 0.4S327 — 0.48099 = 0.0022S. If this difference be divided by the 
amount found necessary for 1 minute it will give the total minutes above 28° 45'; 
0.00228 -^0.000254 = 9. That is, the angle sought is 9 minutes greater than 
28°45 / =28°54 / - 



\CH. I 



MICROSCOPE AND ACCESSORIES 



19 



that this is the sine of 31 45-i-' whence 2 71 or the entire angle (balsam 
or oil angle) is 63 31'. 

That is, three objectives of equal resolving powers, each with a 
numerical aperture of 0.80 would have an angular aperture of 106 16' 
in air, 73 58' in water and 63 31' in homogeneous immersion liquid. 

For the apparatus and method of determining aperture, see 
appendix. 

§ 33. Table of a Group of Objectives with the Numerical 
Aperture (N. A) and the method of obtainiyig it. Half the angular 
aperture is designated by u and the index of refraction of the medhim in 
front of the objective by n. For dry objectives this is air and n = 1, for 
water immersions n = 1.33, and for homogejieous immersions n = 1 .§2 . 
{For a table of natural sines, see third page of cover. ) 



Objective. 3 1! 






< 



25 mm. 

(Dry.) 

25 mm. 
(Dry. ) 

12^ mm. 
(Dry.) 

I2_K mm. 
(Dry.) 

6 mm. 
(Dry.) 

6 mm. 
(Dry. ) 

3 mm. 
(Dry.) 

3 mm. 
(Dry.) 

2 mm. 

Water. 

Immersion. 

2 mm. 

Homogeneous 

Immersion. 



20° 

40° 
42° 

IOO° 

75° 
136° 
115 
163 

no°38 / 



2 mm. 

Homogeneous i34°io / 
Immersion. 



Natural Sine 

of half the angular 

aperture 

(sin u. ) 



Sin = 0. 1736 

2 ,sJ 



r,- 40 

Sin = 0.3420 



Sin ^- = 0.3584 



„ 100 

Sin = 0.7660 



Sin— =0.6087 

o- : 3 6 

Sm — = 0.9272 



Sin ^=0.8434 
Sin — = 0.9890 



n . 96°i2 / 

Sm 2- =0.7443 



Sin 



2 


IIO*^' 


2 


i34°io / 



11 = 1.33 



0.8223 « = 1.52 



n = 1.52 



Index of 
Refraction 
of the medi- 
um in front 
of the objec- 
tive (n). 


Numerical Aperture 
(N. A.) =7zsin u 


11 = I 


N.A.= 


1 X0.1736 = 0.173 


fl = I 


N.A.= 


1 X0.3420 = 0.342 


n= i 


N.A.= 


1 X 0.3583 =0.358 


n = 1 


N.A.= 


1 X 0.7660 = 0.766 


n = 1 


N.A.= 


1 X 0.6087 =0.609 


n = 1 


N. A.= 


1X0.9272 =0.927 


n = 1 


N. A.= 


1X0.8434 = 0.843 


n = 1 


N. A.= 


1X0.9890 = 0.989 



N. A.=i.33X 0.7443 = 0.99 



N. A.= 1.52X0.8223 = 1.25 



N. A.= 1. 52X0. 9210 = 1.40 



20 MICROSCOPE AND ACCESSORIES [CH. I 

§ 34. Significance of Aperture. — As to the real significance of 
aperture in microscopic objectives, it is now an accepted doctrine that — 
the corrections in spherical and chromatic aberration being the same — 
(1) Objectives vary directly as their numerical aperture in their ability 
to define or make clearly visible minute details (resolving power). For 
example an objective of 4 mm. equivalent focus and a numerical aper- 
ture of 0.50 would define or resolve only half as many lines to the 
millimeter or inch as a similar objective of 1.00 N.A. So also an 
objective of 2 mm. focus and 1.40 N.A. would resolve only twice as 
many lines to the millimeter as a 4 mm. objective of 0.70 N.A. Thus 
it is seen that defining power is not a result of magnification but of 
aperture, otherwise the 2 mm. objective would resolve far more than 
twice as man}' lines as the 4 mm. objective. 

Taking the results of the researches of Abbe as a guide to visibility 
with the microscope, one has the general formula 2A.XN.A. That is 
twice the number of wave lengths of the light used multiplied by the 
numerical aperture of the objective. From this general statement it will 
be seen that the shorter the wave lengths of the light, the more there 
will be in an inch or centimeter and therefore the greater the number of 
lines visible in a given space. That is the kind of light used is one ele- 
ment and the objective the other in determining the number of lines 
visible under the microscope. 

Following Mr. E. M. Nelson (Jour. Roy. Micr. Soc, 1893, P- x 5) 
it is believed that not more than ^ths of the numerical aperture .of an 
objective is really available for microscopic stud}-, with a central, solid 
cone of light. To determine the number of lines visible in a given space 
with a given light the formula would become 2A. X Y\ ths N. A. =3/2/tN. A. 
To determine the working-resolving power of any objective it is only 
necessary to know the number of light waves in a given space, say an 
inch or a centimeter and to multiply this number by 3/2 N. A. For 
example suppose one uses ordinary daylight and assumes the average 
wavelength is 146666 in., then there must be 46,666 per inch and 
46,666X3/2 = 70,000 approximately. If the N. A. is 1, then the 
objective will resolve or make visible 70,000 lines to the inch, or ap- 
proximate!}' 28,000 to the centimeter. If blue light were used the 
number would be 32,000 per centimeter, or 80,000 per inch. It will 
be seen that the number of lines here given is smaller than that in the 
table of Carpenter-Dallinger, because in the latter the full aperture 
is supposed to be employed and the light is of the greatest available 
obliquity. 



CH. I] MICROSCOPE AND ACCESSORIES 21 

(2) The illuminating power of an objective of a given focus is 
found to vary directly as the square of the numerical aperture (N. A. ) 2 . 
Thus if two 4 mm. objectives of N.A. 0.20 and N.A. 0.40 were compared 
as to their illuminating power it would be found from the above that 
they would vary as o.20 2 :o.40 2 = 0.0400:0.1600 or 1:4. That is the 
objective of 0.20 N.A. would have but /^th the illuminating power of 
the one of 0.40 N.A. 

(3) The penetrating power, that is the power to see more than one 

plane, is found to van- as the reciprocal of the numerical aperture— — -r- 

so that in an objective of a given focus the greater the aperture the 
less the penetrating power. 

Of course when equivalent focus and numerical aperture both differ 
the problem becomes more complex. 

While all microscopists are agreed that the fineness of detail which 
can be seen depends directly on the numerical aperture of the objective 
used, the general theory of microscopic vision has two interpretations : 

(A.) That it is as with the unaided eye, the telescope and the 
photographic camera. This is the original view and the one which many 
are favoring at the present day (see Mercer, Proceedings of the Amer. 
Micr. Soc. 1896, pp. 321-396). 

(B) The other view originated with Professor Abbe, and in the 
words of Carpenter-Dallinger, pp. 62, 43 : "What this is becomes ex- 
plicable by the researches of Abbe. It is demonstrated that micro- 
scopic vision is sui generis. There is and can be, no comparison between 
microscopic and macroscopic vision. The images of minute objects 
are not delineated microscopically by means of the ordinary laws of 
refraction ; they are not dioptrical results, but depend entirely on the 
laws of diffraction. These come within the scope of and demonstrate 
the undulatory theory of light, and involve a characteristic change 
which material particles or fine structural details, in proportion to their 
minuteness, effect in transmitted rays of light. The change consists 
generally in the breaking up of an incident ray into a group of rays 
with large angular dispersion within the range of which periodic alter- 
nations of dark and light occur. ' ' 

For a consideration of the aperture question, its history and sig- 
nificance, see J. D. Cox, Proc. Amer. Micr. Soc, 1884, pp. 5-39 ; 
Jour. Roy. Micr. Soc, 188 1, pp. 303, 348, 365, 388 ; 1882, pp. 300, 
460; 1883, p. 790; 1884, p. 20 ; 1896, p. 681; 1897, p. 71 JlJjfS^yiPP 1 -, 
354, 362, 592 ; Mercer, Proceedings Amer. Micr. Soc, 1896VPP. 321- 



22 



MICROSCOPE AND ACCESSORIES 



[CH. I 





> 


5f® 


i. 1 




■\W_ 


iff 






$ 1/ 


•^i 


^ c 


/ 


*« 


\ 5 
1\ > 

1 \ ' 


l/j 


^ 


K J \ | 


SIT 


7 


MS 



396 ; Lewis Wright, Philos. Mag., June, 1898, pp. 480-503 ; Carpen- 
ter-Dallinger, Chapter II ; Nelson, Jour. Quekett Micr. Club, VI, pp. 

14-38. 

THE OCULAR 

\ 35. A Microscopic Ocular or Eye-Piece consists of one or more converging 
lenses or lens systems, the combined action of which is, like that of a simple 
microscope, to magnify the real image formed by the objective. 

Fig. 30. Sectional view of a Huygenian ocular to show 
the formation of the Eye-Point. 

Axis .Optic axis of the ocular. D. Diaphragm of the 
ocular. E. L. Eye-Lens. F. L. Field-Lens. 

E. P. Eye-point. As seen tn seclton, it appears some- 
thing like an hour-glass. When seen as looking into the 
ocular, i. e., in transection, it appears as a circle of light. It 
is at the point where the most rays cross. 

Depending upon the relation and action of the different 
lenses forming oculars, they are divided into two great 
groups, negative and. positive. 

§ 36. Negative Oculars are those in which the real, inverted image is formed 
within the ocular, the lower or field-lens serving to collect the image-forming rays 
somewhat, so that the real image is smaller than as if the field-lens were absent 
(Fig. 21). As the field-lens of the ocular aids in the formation of the real image 
it is considered by some to form a part of the objective rather than of the ocular. 
The upper or eye-lens of the ocular magnifies the real image. 

\ 37. Positive Oculars are those in which the real, inverted image of the 
object is formed outside the ocular, and the entire system of ocular lenses magnifies 
the real image like a simple microscope (Fig. 16). 

Positive and negative oculars may be readily distinguished, as a positive ocular 
may be used as a simple microscope, while a negative ocular cannot be so used 
when its field-lens is in the natural position toward the object. By turning the 
eye-lens toward the object and looking into the field-lens an image may be seen, 
however. 

In works and catalogs concerning the microscope and microscopic apparatus, 
and in articles upon the microscope in periodicals, various forms of oculars or eye- 
pieces are so frequently mentioned, without explanation or definition, that it 
seems worth while to give a list, with the French and German equivalents, and a 
brief statement of their character. 

Achromatic Ocular ; Fr. Oculaire achromatique ; Ger. achromatisches Okular. 
Oculars in which chromatic aberration is wholly or nearly eliminated. — Aplanatic 
Ocular ; Fr. Oculaire aplanatique ; Ger. aplanatisches Okular (see § 21). — Binocu- 
lar, stereoscopic Ocular ; Fr. Oculaire binoculaire stereoscopique ; Ger. stereosko- 
pisches Doppel-Okular. An ocular consisting of two oculars about as far apart as 
the two e3-es. These are connected with a single tube which fits a monocular mi- 
croscope. By an arrangement of prisms the image forming rays are divided, half 



CH. /] MICROSCOPE AND ACCESSORIES 23 

being sent to each eye. The most satisfactory form was worked out by Tolles and 
is constructed on true stereotomic principles, both fields being equally illuminated. 
His ocular is also erecting. — CampanVs Ocular (see Huygenian Ocular). — Com- 
pound Ocular ; Fr. Oculaire compose ; Ger. zusammengesetztes Okular. An ocu- 
lar of two or more lenses, e. g., the Huygenian (see Fig. 30). — Continental Ocular. 
An ocular mounted in a tube of uniform diameter as in Fig. 31. — Deep Ocular, 
see high ocular. — Erecting Ocular ; Fr. Oculaire redresseur ; Ger. bildumkeh- 
rendes Okular. An ocular with which an erecting prism is connected so that the 
image is erect as with the simple microscope. Such oculars are most common on 
dissecting microscopes. — Filar micrometer Ocular; Screw m. o., Cobweb m. o., 
Ger. Okular-Schraubenmikrometer. A modification of Ramsden's Telescopic Cob- 
web micrometer ocular. — Goniometer Ocular ; Fr. Oculaire a. goniometre ; Ger. 
Goniometer-Okular. An ocular with goniometer for measuring the angles of minute 
crystals. — High Ocular, sometimes called a deep ocular. One that magnifies 
the real image considerably, i. e., 10 to 20 fold. — Huygenian Ocular, Huygens' O., 
Campani's O., Airy's O.; Fr. Oculaire d'Huygens, o. de Campani ; Ger. Huy- 
gens'sches Okular, Campaniches Okular, see \ 38. — Index Ocular ; Ger. Spitzen- 
O. An ocular with a minute pointer or two pointers at the level of the real image. 
The points are movable and serve for indicators and also, although not satisfac- 
torily, for micrometry. — Kellner's Ocular, see orthoscopic ocular — Low ocular, 
also called shallow ocular. An ocular which magnifies the real image only moder- 
ately, 2. e. , 2 to 8 fold. — Micrometer or micrometric Ocular ; Fr. Oculaire microme- 
trique ou a. micrometre ; Ger. Mikrometer-Okular, Mess Okular, Beneches O., 
Jackson m. o., see \ 4r. — Microscopic Ocular ; Fr. Oculaire microscopique ; Ger. mi- 
kroskopisches Okular. An ocular for the microscope instead of one for a telescope. 
— Negative Ocular, see \ 36. — Nelson's screw-micrometer ocular. A modification of 
the Ramsden's screw or cob-web micrometer in which positive compensating ocu- 
lars may be used. — Orthoscopic Oculars; also called Kellner's Ocular ; Fr. Ocu- 
laire orthoscopique ; Ger. Kellner'sches oder orthoskopisches Okular. An ocular 
with an eye-lens like one of the combinations of an objective (Figs. 22, 23) and a 
double convex field lens. The field-lens is in the focus of the eye-lens and there 
is no diaphragm present. The field is large and flat. — Par-focal Oculars, a series 
of oculars so arranged that the microscope remains in focus when the oculars are 
interchanged (Pennock, Micr. Bulletin, vol. iii, p. 9, 31). — -Periscopic Ocular ; Fr. 
Oculaire periscopique ; Ger. periskopisches Okular. A positive ocular devised by 
Gundlach. It consists of a double convex field-lens and a triplet eye-lens. It 
gives a large, flat field. — Positive Ocular, see \ 37. — Projection Ocular ; Fr. Ocu- 
laire de projection ; Ger. Projections-Okular, see \ 40. — Ramsden's Ocular ; Fr. 
Oculaire de Ramsden ; Ger. Ramsden'sches Okular. A positive ocular devised by 
Ramsden. It consists of two plano-convex lenses placed close together with the 
convex surfaces facing each other. Only the central part of the field is clear. 
Searching Ocular ; Fr. Oculaire d'orientation ; Ger. Sucher-Okular, see § 39, 
Shallow Ocular, see low ocular. — Solid Ocular, holosteric O.; Fr. Oculaire holo- 
stere ; Ger. holosterisches Okular, Vollglass-Okular. A negative eye-piece de- 
vised by Tolles. It consists of a solid piece of glass with a moderate curvature at 
one end for a field-lens, and the other end with a much greater curvature for an 
eye-lens. For a diaphragm, a groove is cut at the proper level and filled with 
black pigment. It is especially excellent where a high ocular is desired. — Spectral 



2 4 



MICROSCOPE AND ACCESSORIES 



\CH. I 



or spectroscopic Ocular ; Fr. Oculaire spectroscopique ; Ger, Spectral-Okular, see 
Microspectroscope, Ch. VI. — Stauroscopic Ocular ; Fr. Oculaire Stauroscopique. 
Ger. Stauroskop-Okular. An ocular with a Bertrand's quartz plate for mineralog- 
ical purposes. — Working Ocular; Fr. Oculaire de travail; Ger. Arbeits-Okular, 
see I 39. 

\ 38. Huygenian Ocular — A negative ocular designed by Huygens for the 
telescope, but adapted also to the microscope. It is the one now most commonly 
emplo)-ed. It consists of a field-lens or collective (Fig. 30), aiding the objective 
in forming the real image, and an eye-lens which magnifies the real image. While 



Ocular Ko 2 




Fig. 31. Compensating Oculars of Zeiss, with section removed to show the con- 
struction. The line A- A is at the level of the upper end of the tube of the micro- 
scope while B-B represents the lower focal points. It will be seen that the mount- 
ing is so arranged that the lower focal points in all are in the same plane and 
therefore the microscope remains in f ones upon changing oculars. ( The oculars are 
par-focal). The lower oculars, 2, 4 and 6 are negative, and the higher ones, 8, 12, 
18, are positive. The numbers 2, 4, 6, 8, 12, iS, indicate the magnification of the 
ocular. From Zeiss' 1 Catalog. ) 

the field-lens aids the objective in the formation of the real, inverted image, and 
increases the field of view, it also combines with the eye-lens in rendering the 
image achromatic. (See §46). 

\ 39. Compensating Oculars. — These are oculars specially constructed for 
use with the apochromatic objectives. They compensate for aberrations outside 
the axis which could not be so readily eliminated in the objective itself. An ocu- 
lar of this kind, magnifying but twice, is made for use with high powers, for the 
sake of the large field in finding objects ; it is called a searching ocular ; those 
ordinarily used for observation are in contradistinction called working oculars. 
Part of the compensating oculars are positive and part negative. (Fig. 31. ) 

\ 40. Projection Oculars. — These are oculars especially designed for project- 
ing a microscopic image on the screen for class demonstrations, or for photo- 
graphing with the microscope. While they are specially adapted for use with 
apochromatic objectives, they may also be used, with ordinary achromatic 
objectives of large numerical aperture. 



CH. /] 



MICROSCOPE AND ACCESSORIES 



25 



Fig. 32. Projection Oculars with section re- 
moved to show the construction. Below are 
shown the upper ends with graduated circle to 
indicate the amount of rotation found necessary 
to focus the diaphragm on the screen. No. 2, 
No. 4. The numbers indicate the amount the 
ocular magnifies the image formed by the 
objective as with the compensation oculars. 
{Zeiss' Catalog.) 

\ 41. Micrometer Ocular. — This is an 
ocular connected with an ocular micrometer. 
The micrometer may be removable, or it may 
be permanently in connection with the ocular, 
and arranged with a spring and screw, by which 
it may be moved back and forth across the 
field. (SeeCh. IV.) 



No. 2 





Fig. 33 Fig. 34 

Figs. 33-34. Ocular Micrometer and movable scale. Fig. jj is a side view of 
the ocular while Fig. J4 gives a sectional end view, and shows the ocular micrometer 
in position, hi both the screw which moves the micrometer is shown at the left. 
( From Bausch & Lomb Opt. Co. ) 

§ 42. Spectral or Spectroscopic Ocular. — (See Micro-Spectroscope, Ch. VI). 
DESIGNATION OP OCULARS 



, \ 43. Equivalent Focus. — As with objectives, some opticians designate the 
oculars by their equivalent focus ( \ 15 ). With this method the power of the ocular, 
as with objectives, varies inversely as the equivalent focal length, and therefore 
the greater the equivalent focal length the less the magnification. This seems as 
desirable a mode for oculars as for objectives and is coming more and more into 
use by the most progressive opticians. It is the method of designation advo- 
cated by Dr. R. H. Ward for many years, and was recommended by the committee 
of the American Microscopical Society, (Proc. Amer. Micr. Soc, 1883, p. 175, 1884, 
p. 228). 



26 



MICROSCOPE AND ACCESSORIES 



\_CH.I 




Fig. 35. Ocular Screw- Micrometer with 
compensation ocular 6. The upper figure 
shows a sectional view of the ocular and the 
screw for moving the micrometer at the right. 
At the left is shown a clamping screw to 
fasten the ocular to the upper part of the mi- 
croscope hibe. Below is a face view, showing 
the graduation on the wheel. An ocular 
micrometer like this is in general like the 
cob-web micrometer and may be used for 
measuring objects of varying sizes very accu- 
rately. With the oj'dinary ocular micrometer 
jLgflilf /fc"™ , ijfc\ 1 I gSLr? te^l very small objects frequently fill but a part of 
- * V fB^m^f / e BB g^ nyp^ll an interval of the micrometer, but with this 

the movable cross lines traverse the object (or 
rather its real image') regardless of the minute- 
ness of the object. (Zeiss' Catalog). 
I 44. Numbering and Lettering. — Oculars like objectives may be numbered or 
lettered arbitrarily. When so designated, the smaller the number, or the earlier 
the letter in the alphabet, the lower the power of the ocular. 

\ 45. Magnification. — The compensating oculars are marked with the amount 
they magnify the real image. Thus an ocular marked X 4> indicates that the real 
image of the objective is magnified four fold by the ocular. 

The projection oculars are designated simply by the amount the}' multiply the 
real image of the objective. Thus for the short or 160 mm. tube-length they are, 
X2, X4 ; and for the long or 250 mm. tube, they are X3 an d X6. That is, the 
final image on the screen or the ground glass of the photographic camera will be 
2, 3, 4, or 6 times greater than it would be if no ocular were used. See Ch. VIII. 
\ 46. Standard Size Oculars. — The Royal Microscopical Society of London 
took a very important step (Dec. 20, 1S99) in establishing standard sizes for ocu- 
lars and sub-stage condensers. To quote from the Journal of the Royal Micro- 
scopical Society for 1900, p. 147 : 

Resolved, "That the standard size for the inside diameter of the substage fit- 
ting be 1.527 in. =3S. 786 mm. That tbe gauges for standardizing eye-pieces be 
the internal diameters of the draw-tubes, the tightness of the fit being left to the 
discretion of the manufacturers. ' ' 

The sizes for oculars are four in number, 1 and 2 being most common. 

( 1 ) 0.9173 inch = 23.300 mm. This is the Continental size. 

(2) 1.04 inch = 26.416 mm. This is the size used by the English Opticians 

for student and small microscopes. 

Medium size binoculars (English.) 
Long tube binoculars. 
For the history of the Huygenian Ocular, and a discussion of formulae for its 
construction, see Nelson, J. R. M. S., 1900, p. 162-169. 

EXPERIMENTS 

§ 47. Putting an Objective in Position and Removing it. — 
Elevate the tube of the microscope by means of the coarse adjustment 



(3) 
(4) 



1.27 
1. 41 



inch =32.258 mm. 
inch = 35.814 mm. 



CH. /] 



MICROSCOPE AND ACCESSORIES 



27 



(frontispiece) so that there may be plenty of room between its lower 
end and the stage. Grasp the objective lightly near its lower end with 
two fingers of the left hand, and hold it against the nut at the lower 
end of the tube. With two fingers of the right hand take hold of the 
milled ring near the back or upper end of the objective and screw it 
into the tube of the microscope. Reverse this operation for removing 
the objective. By following this method the danger of dropping the 
objective will be avoided. 

§ 4S. Putting an Ocular in Position and Removing it. — Ele- 
vate the body of the microscope with the coarse adjustment so that the 
objective will be 2 cm. or more from the object — grasp the ocular by 
the milled ring next the eye-lens (Fig. 21), and the coarse adjustment 
or the tube of the microscope and gently force the ocular into position. 
In removing the ocular, reverse the operation. If the above precau- 
tions are not taken, and the oculars fit snugly, there is danger in in- 
serting them of forcing the tube of the microscope downward and the 
objective upon the object. 

§ 49. Putting an Object under the Microscope. — This is so 
placing an object under the simple microscope, or on the stage of the 
compound microscope, that it will be in the field of view when the 
microscope is in focus (§ 50). 

With low powers, it is not difficult to get an object under the 
microscope. The difficulty increases, however, with the power of the 
microscope and the smallness of the object. It is usually necessary to 
move the object in various directions while looking into the micro- 
scope, in order to get it into the field. Time is usually saved by get- 
ting the object in the center of the field with a low objective before 
putting the high objective in position. This is greatly facilitated by 
using a nose-piece, or revolver. (See Figs. 36~36a, and the pictures of 
microscopes, Ch. II.) 





Fig. 36. Triple nose-piece or revol- 
ver for quickly changing objectives. ( The 
Spencer Lens Co. ) 



Fig. 36a. Triple nose-piece or re- 
volver for quickly changing objectives. 
( The Bausch & Lomb Optical Co. ) 



28 MICROSCOPE AND ACCESSORIES [CH. I 

§ 50. Field or Field of View of a Microscope. — This is the 
area visible through a microscope when it is in focus. When properly 
lighted and there is no object under the microscope, the field appears 
as a circle of light. When examining an object it appears within the 
light circle, and by moving the object, if it is of sufficient size, differ- 
ent parts are brought successively into the field of view. 

In general, the greater the magnification of the entire microscope, 
whether the magnification is produced mainly by the objective, the 
ocular, or by increasing the tube length, or by a combination of all 
three (see Ch. IV, under magnification), the smaller is the field. 

The size of the field is also dependent, in part, without regard to 
magnification, upon the size of the opening in the ocular diaphragm. 
Some oculars, as the orthoscopic and periscopic, are so constructed as 
to eliminate the ocular diaphragm, and in consequence, although this 
is not the sole cause, the field is considerably increased. The exact 
size of the field may be read off directly by putting a stage micrometer 
under the microscope and noting the number of spaces required to 
measure the diameter of the light circle. 




17 mm 



Fig. 37. Figures showing approximately the actual size of the field with ob- 
jectives 0/85 mm., 45 mm., ij mm., 5 mm., and 2 mm., equivalent focus, and 
ocular of 37 y z nun., equivalent focus in each case. This figure shows graphically 
what is also very clearly indicated in the table (| 52). 

§ 51. The size of the field of the microscope as projected into the 
field of vision of the normal human e}*e (z. e. , the virtual image) may 
be determined by the use of the camera lucida with the drawing surface 
placed at the standard distance of 250 millimeters (Ch. IV.) 

§ 52. Table showing the actual size in millimeters of the field of a 
group of commonly used objectives and oculars. Compare with the graphic 
representation in Fig. jj . See also § 50. 



CH. /] 



MICROSCOPE AND ACCESSORIES 



29 



I Equivalent 
Focus and 
N. A. of 
Objective 


Diameter 
of Field 
in mm. 


Equivalent 

Focus of 

Ocular 


Kind of 
Ocular 


85 mm 


15-4 
10.6 

3-3 


37K mm. 
25 
12K " 


Huygenian 


45 mm 


7.0 

5-o 
4.0 


37^ mm. 
25 

I2>< " 


Huygenian 


17 mm. . 
N. A. =0.25 


3-0 
2.0 
1.6 


37^ mm. 
25 
12K " 


Huygenian 


5-7 

2.8 

1.4 
o.97 


180 mm. 
45 
15 
10 " 


Compensation 


5 mm. . .. 
N. A. =0.92 


o.54i 
0.371 
0.290 


37^ mm. 
25 
1234 " 


Huygenian 


0.S50 
0.501 
0.250 
O.I73 


180 mm. 
45 
15 
10 " 


Compensation 


2 mm. . 

N. A. = 1.25 



0.270 
0.186 
0.147 


37K mm. 
25 
12^ " 


Huygenian 


0.450 
0.251 
0.125 
0.088 


180 mm. 
45 
15 
10 " 


Compensation 



FUNCTION OF AN OBJECTIVE 

§ 53. Put a 50 mm. objective on the microscope or screw off the 
front combination of a 16 mm.,(^3-in.), and put the back combination 
on the microscope for a low objective. 

Place some printed letters or figures under the microscope, and 
light well. In place of an ocular put a screen of ground glass, or a 
piece of lens paper, over the upper end of the tube of the microscope.* 

*Ground glass may be very easily prepared by placing some fine emery or 
carborundum between two pieces of glass, wetting it with water and then rubbing 
the glasses together for a few minutes. If the glass becomes too opaque, it may 
be rendered more translucent by rubbing some oil upon it. 



3© MICROSCOPE AND ACCESSORIES [CH. I 

Lower the tube of the microscope by means of the coarse adjust- 
ment until the objective is within 2-3 cm. of the object on the stage. 
Look at the screen on the top of the tube, holding the head about as 
far from it as for ordinary reading, and slowly elevate the tube b3 T means 
of the coarse adjustment until the image of the letter appears on the 
screen. 

The image can be more clearly seen if the object is in a strong 
light and the screen in a moderate light, i. e. , if the top of the micro- 
scope is shaded. 

The letters will appear as if printed on the ground glass or paper, 
but will be inverted (Fig. 21). 

If the objective is not raised sufficiently, and the head is held too 
near the microscope, the objective will act as a simple microscope. If 
the letters are erect, and appear to be down in the microscope and not 
on the screen, hold the head farther from it, shade the screen, and 
raise the tube of the microscope until the letters do appear on the 
ground glass. 

To demonstrate that the object must be outside the principal focus 
with the compound microscope, remove the screen and turn the tube of 
the microscope directly toward the sun. Move the tube of the micro- 
scope with the coarse adjustment until the burning or focal point is 
found (§ 6). Measure the distance from the paper object on the stage 
to the objective, and it will represent approximately the principal 
focal distance (Figs. 10, 11). Replace the screen over the top of the 
tube, no image can be seen. Slowly raise the tube of the microscope 
and the image will finally appear. If the distance between the object 
and the objective is now taken, it will be found considerably greater 
than the principal focal distance (compare § n). 

§ 54. Aerial Image. — After seeing the real image on the ground- 
glass, or paper, use the lens paper over about half of the opening of 
the tube of the microscope. Hold the eye about 250 mm. from the 
microscope as before and shade the top of the tube by holding the hand 
between it and the light, or in some other way. The real image can 
be seen in part as if on the paper and in part in the air. Move the 
paper so that the image of half a letter will be on the paper and half 
in the air. Another striking experiment is to have a small hole in the 
paper placed over the center of the tube opening, then if a printed word 
extends entirely across the diameter of the tube its central part may be 
seen in the air, the lateral parts on the paper. The advantage of the 
paper over part of the opening is to enable one to accommodate the 



CH.I~\ MICROSCOPE AND ACCESSORIES 31 

eyes for the right distance. If the paper is absent the eyes adjust 
themselves for the light circle at the back of the objective, and the 
aerial image appears low in the tube. Furthermore it is more difficult 
to see the aerial image in space than to see the image on the ground- 
glass or paper, for the eye must be held in the right position to receive 
the rays projected from the real image, while the granular surface of 
the glass and the delicate fibres of the paper reflect the raj's irregularly, 
so that the image ma\- be seen at almost an} 7 angle, as if the letters 
were actually printed on the paper or glass. 

§ 55. The Function of an Objective, as seen from these experi- 
ments, is to form an enlarged, inverted, real image of an object, this 
image being formed on the opposite side of the objective from the 
object (Fig. 21). 

FUNCTION OF AN OCULAR 

§ 56. Using the same objective as for § 53, get as clear an image 
of the letters as possible on the lens paper screen. Look at the image 
with a simple microscope (Fig. 17 or 18) as if the image were an object. 

Observe that the image seen through the simple microscope is 
merely an enlargement of the one on the screen, and that the letters 
remain inverted, that is they appear as with the naked eye (§ 11). 
Remove the screen and observe the aerial image with the tripod. 

Put a 50 mm. (A, No. 1 or 2 in.), ocular i. e., an ocular of low 
magnification) in position (§ 48). Hold the eye about 10 to 20 milli- 
meters from the eye-lens and look into the microscope. The letters 
will appear as when the simple microscope was used (see above), the 
image will become more distinct by slightly raising the tube of the 
microscope with the coarse adjustment. 

§ 57. The Function of the Ocular, as seen from the above, is 
that of a simple microscope, viz. : It magnifies the real image formed 
by the objective as if that image were an object. Compare the image 
formed by the ocular (Fig. 21), and that formed by a simple microscope 
(Fig. 38). 

It should be borne in mind, however, that the rays from an object 
as usually examined with a simple microscope, extend from the object 
in all directions, and no matter at what angle the simple microscope is 
held, provided it is sufficiently near and points toward the object, an 
image may be seen. The rays from a real image, however, are continued 
in certain definite lines and not in all directions ; hence, in order 
to see this aerial image with an ocular or simple microscope, or 



32 



MICROSCOPE AND ACCESSORIES 



\CH. I 



in order to see the aerial image with the unaided eye, the simple micro- 
scope, ocular or eye must be in the path of the rays (Fig. 21.) 

Fig. 38. Diagram of the simple microscope 
showing the course of the rays and all the images, 
and that the eye forms an integral part of it. 

A 1 B 1 . The object within the principal focus . 
A= B 3 . The virtual image on the same side of 
the lens as the object. It is indicated by dotted 
lines, as it has no actual existence. 

B- A 2 . Retinal image of the object (A 1 B 1 ). 
The virtual image is simply a projection of the 
retinal image into the field of vision. 

Axis. The principal optic axis of the micro- 
scope and of the eye. Cr. Cornea of the eye. L. 
Crystalline lens of the eye. R. Ideal refracting 
surf ace at which all the refractions of the eye may 
be assumed to take place. 

•'-i-i J 2? 




§ 58. The field-lens of a Huygenian ocular makes the real 
image smaller and consequently increases the size of the field ; it also 
makes the image brighter by contracting the area of the real image. 
(Fig. 30.) Demonstrate this by screwing off the field-lens and using 
the eye-lens alone as an ocular, refocusing if necessary. Note also that 
the image is bordered by a colored haze (§7). 

When looking into the ocular with the field-lens removed, the eye 
should not be held so close to the ocular, as the eye-point is consider- 
ably farther away than when the field-lens is in place. 

§ 59. The eye-point. — This is the point above the ocular or 
simple microscope where the greatest number of emerging rays cross. 
Seen in profile, it may be likened to the narrowest part of an hour 
glass. Seen in section (Fig. 30), it is the smallest and brightest 
light circle above the ocular. This is called the eye-point, for if the 
pupil of the eye is placed at this level, it will receive the greatest 
number of rays from the microscope, and consequently see the largest 
field. 

Demonstrate the eye-point by having in position an objective and 
ocular as above (§ 53). Light the object brightly, focus the micro- 
scope, shade the ocular, then hold some ground-glass or a piece of the 
lens paper above the ocular and slowly raise and lower it until the 
smallest circle of light is found. By using different oculars it will be 



CH. /.] MICROSCOPE AND ACCESSORIES 33 

seen that the eye-point is nearer the eye-lens in high than in low ocu- 
lars, that is the eye-point is nearer the eye-lens for an ocular of small 
equivalent focus than for one of greater focal length. 

REFERENCES FOR CHAPTER I 

In chapter X will be given a bibliography, with full titles, of the works and 
periodicals referred to. 

For the subjects considered in this chapter, general works on the microscope 
may be consulted with great advantage for different or more exhaustive treatment. 
The most satisfactory work in English is Carpenter-Dallinger, 8th Ed. For the 
history of the microscope, Mayall's Cantor Lectures on the microscope are very 
satisfactory. For a continuation of the history begun by Mayall in the Cantor 
Lectures see Nelson, Journal of the Oueckett Micr. Club, and the Jour. Roy. 
Micr. Soc, 1S97-1901— . Carpenter-Dallinger, 8th Ed. Petri, Das Mikroskop. 

The following special articles in periodicals may be examined with advantage : 

Apochromatic Objectives, etc. Dippel in Zeit. wiss. Mikr., 1886, p. 303 ; also 
in the Jour. Roy. Micr. Soc, 1S86, pp. 316, 849, 11 10, ; same, 1890, p. 480 ; Zeit. f. 
Instrumentenk., 1890, pp. 1-6 ; Micr. Built., 1891, pp. 6-7. 

Tube-length, etc. Gage, Proc. Amer. Soc. Micrs., 1887, pp. 168-172 ; also in 
the Microscope, the Jour. Roy. Micr. Soc, and in Zeit, wiss. Mikr., 1887-8. 
Bausch, Proc. Amer. Soc. Micrs., 1890, pp. 43-49 ; also in the Microscope, 1890, 
pp. 2S9-296. 

Aperture. J. D. Cox, Presidential Address, Proc. Amer. Soc. Micrs., 1884, pp, 
5-39, Jour. Roy. Micr. Soc, 1881, pp. 303, 348, 365, 388 ; 1882, pp. 300, 460 ; 1883, 
p. 790 ; 1884, p. 20. Czapski, Theorie der optischen Instrumente nach Abbe. 
See also references in \ 34. 



The Barnes Dissecting Microscope ( The Bausch & Lomb Optical Company) . 



CHAPTER II 



LIGHTING AND FOCUSING; MANIPULATION OF DRY, 

ADJUSTABLE AND IMMERSION OBJECTIVES ; CARE 

OF THE MICROSCOPE AND OF THE EYES ; 

LABORATORY MICROSCOPES 



APPARATUS AND MATERIAL FOR THIS CHAPTER 

Microscope supplied with plane and concave mirror, Achromatic and Abbe 
condensers, dry, adjustable and immersion objectives, oculars, triple nose-piece. 
Microscope lamp and movable condenser (bull's eye or other form, Fig. 53), 
Homogeneous immersion liquid ; Benzin, alcohol, distilled water ; Mounted prep- 
aration of fly's wing(§ 70); Mounted preparation of Pleitrosigma ($ 77, 78). Stage 
or ocular micrometer ($ 92); Glass slides and cover-glasses (Ch. VII); 10 per ct. 
solution of salicylic acid in 95 per ct. alcohol ( \ 92 ) ; Preparation of stained bac- 
teria (\ 108); Vial of equal parts olive or cotton' seed oil or liquid vaselin and 
benzin ($ 112): Double eye shade (Fig. 60); Screen for whole microscope (Fig. 59). 

FOCUSING 

\ 60. Focusing is mutually arranging an object and the microscope so that a 
clear image may be seen. 

With a simple microscope (| 11) either the object or the microscope or both 
may be moved in order to see the image clearly, but with the compound microscope 
the object more conveniently remains stationary on the stage, and the tube or 
body of the microscope is raised or lowered (frontispiece). 

In general, the higher the power of the whole microscope whether simple or 
compound, the nearer together must the object and objective be brought. With 
the compound microscope, the higher the objective, and the longer the tube of 
the microscope, the nearer together must the object and the objective be brought. 
If the oculars are not par-focal, the higher the magnification of the ocular, the 
nearer must the object and objective be brought. 

\ 61. Working Distance. — By this is meant the space between the simple mi- 
croscope and the object, or between the front lens of the compound microscope and 
the object, when the microscope is in focus. This working distance is always con- 
siderably less than the equivalent focal length of the objective. For example, 
the front-lens of a 6 mm. or J/jfth in. objective would not be 6 millimeters or Xth 
inch from the object when the microscope is in focus, but considerably less than 
that distance. If there were no other reason than the limited working distance of 
high objectives, it would be necessary 7 to use a very thin cover-glass over the 



CH. II] 



LIGHTING AND FOCUSING 



35 



object. (See § 24, 29.) If too thick covers are used it may be impossible to get 
an objective near enough an object to get it in focus. For objects that admit of 
examination with high powers it is always better to use thin covers. 

LIGHTING WITH DAYLIGHT 

§ 62. Unmodified sunlight should not be employed except in special cases. 
North light is best and most uniform. When the sky is covered with white clouds 
the light is most favorable. To avoid the shadows produced by the hands in 
manipulating the mirror, etc., it is better to face the light ; but to protect the 
eyes and to shade the stage of the microscope some kind of screen should be 
used. The one figured in (Fig. 62) is cheap and efficient. If one dislikes to face 
the window or lamp it is better to sit so that the light will come from the left as in 
reading. 

It is of the greatest importance and advantage for one who is to use the 
microscope for serious work that he should comprehend and appreciate thoroughly 
the various methods of illumination, and the special appearances due to different 
kinds of illumination. 

Depending on whether the light illuminating an object traverses the object or 
is reflected upon it, and also whether the object is symmetrically lighted, or 
lighted more on one side than the other, light used in microscopy is designated as 
reflected and transmitted ', axial and oblique. 




Sta.g 




39 
Figs. 39-40. For full explanation see Figs. 22 and 23. 



40 



\ 63. Reflected, Incident or Direct Light. — By this is meant light reflected 
upon the object in some way and then irregularly reflected from the object to the 
microscope. By this kind of light objects are ordinarily seen by the unaided eye, 
and the objects are mostly opaque. In Vertebrate Histology, reflected light is but 



36 LIGHTING AND FOCUSING [CH. II 

little used ; but in the study of opaque objects, like whole insects, etc., it is used 
a gfeat deal. For low powers, ordinary daylight that naturally falls upon the 
object, or is reflected or condensed upon it with a mirror or condensing lens, 
answers very well. For high powers and for special purposes, special illuminating 
apparatus has been devised (§ 28). (See also Carpenter-Dallinger, Ch. IV). 

§ 64. Transmitted Light. — By this is meant light which passes through an 
object from the opposite side. The details of a photographic negative are in 
many cases only seen or best seen by transmitted light, while the print made from 
it is best seen by reflected light. 

Almost all objects studied in Vertebrate Histology are lighted by transmitted 
light, and they are in some way rendered transparent or semi-transparent. The 
light traversing and serving to illuminate the object in working with a compound 
microscope is usually reflected from a plane or concave mirror, or from a mirror to 
a condenser ($88), and thence transmitted to the object from below (Figs. 48-51). 

I 65. Axial or Central Light. — By this is understood light reaching the object, 
the rays of light being parallel to each other and to the optic axis of the micro- 
scope, or a diverging or converging cone of light whose axial ray is coincident with 
the optic axis of the microscope. In either case the object is symmetrically 
illuminated. 

I 66. Oblique Light. — This is light in which parallel ra5^s from a plane mirror 
form an angle with the optic axis of the microscope (Fig. 40). Or if a concave 
mirror or a condenser is used, the light is oblique when the axial ray of the cone 
of light forms an angle with the optic axis (Fig. 40). 

DIAPHRAGMS 

\ 67. Diaphragms and their Proper Employment. — Diaphragms are opaque 
disks with openings of various sizes, which are placed between the source of light 
or mirror and the object. In some cases an iris diaphragm is used, and then the 
same one is capable of giving a large range of openings. The object of a dia- 
phragm in general, is to cut off all adventitious light and thus enable one to light 
the object in such a way that the light finally reaching the microscope shall all 
come from the object or its immediate vicinity. The diaphragms of a condenser 
serve to vary its aperture to the needs of each object and each objective. 

\ 68. Size and Position of Diaphragm Opening. — When no condenser is used 
the size of the opening in the diaphragm should be about that of the front lens 
of the objective. For some objects and some objectives this rule may be quite 
widely departed from ; one must learn by trial. 

When lighting with a mirror the diaphragm should be as close as possible to 
the object in order, (a) that it may exclude all adventitious light from the object ; 
(b) that it may not interfere with the most efficient illumination from the mirror 
by cutting off a part of the illuminating pencil. If the diaphragm is a considera- 
ble distance below the object, ( 1 ) it allows considerable adventitious light to reach 
the object and thus injures the distinctness of the microscope image ; (2) it pre- 
vents the use of very oblique light unless it swings with the mirror ; ( 3 ) it cuts off 
a part of the illuminating cone from a concave mirror. On the other hand, even 
with a small diaphragm, the whole field will be lighted. 



CH. II] LIGHTING AND FOCUSING 37 

With an illuminator or condenser (Figs. 41 , 48) , the diaphragm serves to narrow 
the pencil to be transmitted through the condenser, and thus to limit the aperture 
(see \ 84). Furthermore, by making the diaphragm opening eccentric, oblique 
light may be used, or by using a diaphragm with a slit around the edge (central 
stop diaphragm), the center remaining opaque, the object may be lighted with a 
hollow cone of light, all of the rays having great obliquity. In this way the so- 
called dark-ground illumination may be produced (§ 92 ; Fig. 51). 

ARTIFICIAL ILLUMINATION 

\ 69. For evening work and for certain special purposes, artificial illumina- 
tion is employed. A good petroleum (kerosene) lamp with flat wick has been 
found very satisfactory, also an incandescent electric or Welsbach light, but for 
brilliancy and for the actinic power necessary for very rapid photo-micrography 
(see Ch. VIII) the electric arc lamp or an acetylene lamp serves well. Whatever 
source of artificial light is employed, the light should be brilliant and steady. 

LIGHTING AND FOCUSING : EXPERIMENTS 

§ 70. Lighting with a Mirror. — As the following experiments 
are for mirror lighting only, remove the substage condenser if present 
(see § 79, for condenser). Place a mounted fly's wing under the 
microscope, put the 16 mm.(/3 in.) or other low objective in position, 
also a low ocular. With the coarse adjustment lower the tube of the 
microscope to within about 1 cm. of the object. Use an opening in 
the diaphragm about as large as the front lens of the objective ; 
then with the plane mirror try to reflect light up through the diaphragm 
upon the object. One can tell when the field (§ 50) is illuminated, by 
looking at the object on the stage, but more satisfactorily by looking 
into the microscope. It sometimes requires considerable manipulation 
to light the field well. After using the plane side of the mirror turn 
the concave side into position and light the field with it. As the con- 
cave mirror condenses the light, the field will look brighter with it than 
with the plane mirror. It is especially desirable to remember that the 
excellence of lighting depends in part on the position of the diaphragm 
(§ 68). If the greatest illumination is to be obtained from the concave 
mirror, its position must be such that its focus will be at the level of 
the object. This distance can be very easily determined by finding the 
focal point of the mirror in full sunlight. 

§ 71. Use of the Plane and of the Concave Mirror. — The mir- 
ror should be freely movable, and have a plane and a concave face. The 
concave face is used when a large amount of light is needed, the plane 
face when a moderate amount is needed or when it is necessary to have 
parallel rays or to know the direction of the rays. 



38 LIGHTING AND FOCUSING [CH. II 

§ 72. Focusing with Low Objectives. — Place a mounted fly's 
wing under the microscope ; put the 16 mm. (fi in.) objective in 
position, and also the lowest ocular. Select the proper opening in the 
diaphragm and light the object well with transmitted light (§ 64, 68). 

Hold the head at about the level of the stage, look toward the 
window, and between the object and the front of the objective ; with 
the coarse adjustment lower the tube until the objective is within 
about half a centimeter of the object. Then look into the microscope 
and slowly elevate the tube with the coarse adjustment. The image 
will appear dimly at first, but will become very distinct by raising the 
tube still higher. If the tube is raised too high the image will become 
indistinct, and finally disappear. It will again appear if the tube is 
lowered the proper distance. 

When the microscope is well focused try both the concave and the 
plane mirrors in various positions and note the effect. Put a high 
ocular in place of the low one (§43). If the oculars are not par- 
focal it will be necessary to lower the tube somewhat to get the micro- 
scope in focus.* 

Pull out the draw-tube 4-6 cm., thus lengthening the body of the 
microscope ; it will be found necessary to lower the tube of the micro- 
scope somewhat. (For reason, see Fig. 58.) 

§ 73. Pushing in the Draw-Tube. — To push in the draw-tube, 
grasp the large milled ring of the ocular with one hand, and the 
milled head of the coarse adjustment with the other, and gradually 
push the draw-tube into the tube. If this were done without these 
precautions the objective might be forced against the object and the 
ocular thrown out by the compressed air. 

§ 74. Focusing with High Objectives. — Employ the same 
object as before, elevate the tube of the microscope and, if no revolving 
nose-piece is present, remove the 16 mm. (73 in.) objective as indi- 
cated. Put the 3 mm. (}& in.) or a higher objective in place, and use 
a low ocular. 



* Par-focal oculars are so constructed, or so mounted, that those of different 
powers may be interchanged without the microscopic image becoming wholly out 
of focus (Fig. 31). When high objectives are used, while the image may be 
seen after changing oculars, the instrument nearly always needs slight focusing. 
With low powers this may not be necessary. 

Objectives are also now commonly mounted in the triple or double revolving 
nose-pieces (,Figs. 36, 36 a) so that if one of the objectives is in focus either of the 
others will be approximately, in focus when turned into position. This is a very 
great convenience. 



CH. II '] LIGHTING AND FOCUSING 39 

Light well, and employ the proper opening in the diaphragm, etc. 
(§ 6S). Look between the front of the objective and the object as 
before (§72), and lower the tube with the coarse adjustment till the 
objective almost touches the cover-glass over the object. Look into 
the microscope, and with the coarse adjustment, raise the tube very 
slowly until the image begins to appear, then turn the milled head of 
the fine adjustment (frontispiece), first one way and then the other, if 
necessary, until the image is sharply defined. 

In practice it is found of great advantage to move the preparation 
slightly while focusing. This enables one to determine the approach 
to the focal point either from the shadow or the color, if the object is col- 
ored. With high powers and scattered objects there might be no object 
in the small field (see § 50, Fig. 37 for size of field). By moving the 
preparation an object will be moved across the field and its shadow 
gives one the hint that the objective is approaching the focal point. It 
is sometimes desirable to focus on the edge of the cement ring or on 
the little ring made by the marker (see Figs. 61-66). 

Xote that this high objective must be brought nearer the object 
than the low one, and that by changing to a higher ocular (if the ocu- 
lars are not par-focal) or lengthening the tube of the microscope it 
will be found necessary to bring the objective still nearer the object, as 
with the low objective. (For reason see Fig. 58.) 

§ 75. Always Focus Up, as directed above. If one lowers the 
tube only when looking at the end of the objective as directed above, 
there will be no danger of bringing the objective in contact with the 
object, as may be done if one looks into the microscope and focuses 
down. 

When the instrument is well focused, move the object around in 
order to bring different parts into the field. It may be necessary to 
re-focus with the fine adjustment every time a different part is brought 
into the field. In practical work one hand is kept on the fine adjust- 
ment constantly, and the focus is continually varied. 

§ 76. Determination of Working Distance. — As stated in § 61, 
this is the distance between the front lens of the objective and the 
object when the objective is in focus. It is always less than the equiv- 
lent focal length of the objective. 

Make a wooden wedge 10 cm. long which shall be exceedingly thin 
at one end and about 20 mm. thick at the other. Place a slide on the 
stage and some dust on the slide. Do not use a cover-glass. Focus the 
dust carefully first with the low then with the high objective. When 



4-0 LIGHTING AND FOCUSING [CH. II 

the objective is in focus push the wedge under the objective on the 
slide until it touches the objective. Mark the place of contact with a 
pencil and then measure the thickness of the wedge with a rule 
opposite the point of contact. This thickness will represent very 
closely the working distance. For measuring the thickness of the 
wedge at the point of contact for the high objective use a steel scale 
ruled in iths mm. and the tripod to see the divisions. Or one may 
use a cover-glass measure (Ch. VIII; for determining the thickness of 
the wedge. 

For the higher powers if one has a microscope in which the fine ad- 
justment is graduated, the working distance may be readily determined 
when the thickness of the cover-glass over the specimen is known, as 
follows : Get the object in focus, lower the tube of the microscope, un- 
til the front of the objective just touches the cover-glass. Note the 
position of the micrometer screw and slowly focus up with the fine 
adjustment until the object is in focus. The distance the objective was 
raised plus the thickness of the cover-glass represents the working dis- 
tance. For example, a 3 mm. objective after being brought in contact 
with the cover-glass was raised by the fine adjustment a distance repre- 
sented by 16 of the divisions on the head of the micrometer screw. 
Each division represented .01 mm., consequently the objective was 
raised . 16 mm. As the cover-glass on the specimen used was . 15 mm. 
the total working distance is .16 -r.15 =.31 mm. 

\ 76a. Free Working Distance. — In the microscope catalog of Zeiss there is 
given a table of the size of the field and also of the "free working-distance." This 
free working-distance is the space between the lower end of the objective and the 
cover glass of y 1 ^ mm. thickness, when the objective is in focus on an object imme- 
diately under the cover. This is exceedingly practical information for a possessor 
of a microscope, and it is hoped that the other opticians will adopt the suggestion. 
Naturally, however, the free working-distance for each optician should be reckoned 
from the top of the cover for which his unadjustable objectives are corrected. If, 
for example, the thickness of cover for which an objective is corrected is -^ mm. 
then the free working-distance should be that between the top of this and the 
objective when the objective is in focus on an object under the cover. (See the 
table of cover-glass thickness, p. 14). 

CENTRAL AND OBLIQUE LIGHT WITH A MIRROR 

§ 77. Axial or Central Light (§ 65). — Remove the condenser 
or any diaphragm from the substage, then place a preparation contain- 
ing minute air bubbles under the microscope. The preparation may 
be easily made by beating a drop of mucilage on a slide and covering 



CH. 1I~\ LIGHTING AND FOCUSING 41 

it (see Ch. Ill) . Use a 3 mm. , ( }i in. ) or No. 7 objective and a medium 
ocular. Focus the microscope and select a very small bubble, one 
whose image appears about 1 mm. in diameter, then arrange the plane 
mirror so that the light spot in the bubble appears exactly in the 
center. Without changing the position of the mirror in the least, 
replace the air bubble preparation by one of Pleurosigma angulatum or 
some other finely marked diatom. Stud}' the appearance very carefully. 

§ 78. Oblique Light (§66). — Swing the mirror far to one side 
so that the rays reaching the object may be very oblique to the optic 
axis of the microscope. Study carefully the appearance of the diatom 
with the oblique light. Compare the appearance with that where central 
light is used. The effect of oblique light is not so striking with histo- 
logical preparations as with diatoms. 

It should be especially noted in §§ 77, 78, that one cannot deter- 
mine the exact direction of the rays by the position of the mirror. 
This is especially true for axial light ( §77). To be certain the light 
is axial some such test as that given in § 77 should be applied. (See 
also Ch. Ill, under Air-bubbles.) 

CONDENSERS OR ILLUMINATORS* 

§ 79. These are lenses or lens-systems for the purpose of illuminat- 
ing with transmitted light the object to be studied with the microscope. 

For the highest kind of investigation their value cannot be over- 
estimated. They may be used either with natural or artificial light, 
and should be of sufficient numerical aperture to satisfy objectives of 
the widest angle. 



*No one has stated more cleat ly, or appreciated more truly the value of cor- 
rect illumination and the methods of obtaining it than Sir David Brewster, 1820, 
183 1. He says of illumination in general : "The art of illuminating microscopic 
objects is not of less importance than that of preparing them for observation. " 
"The eye should be protected from all extraneous light, and should not receive any 
of the light which proceeds from the illuminating center, excepting that portion 
of it which is transmitted through or reflected from the object." So likewise the 
value and character of the substage condenser was thoroughly understood and 
pointed out by him as follows : "I have no hesitation in saying that the apparatus 
for illumination requires to be as perfect as the apparatus for vision, and on this 
account I would recommend that the illuminating lens should be perfectly free of 
chromatic and spherical aberration, and the greatest care be taken to exclude all 
extraneous light both from the object and from the eye of the observer." See Sir 
David Brewster's treatise on the Microscope, 1837, pp. 136, 138, 146, and the 
Edinburgh Journal of Science, new series, No. 11 (1831) p. 83. 



42 LIGHTING AND FOCUSING [CH. II 

It is of the greatest advantage to have the sub-stage condenser 
mounted so that it may be easily moved up or down under the stage. 
The iris diaphragm is so convenient that it should be furnished in all 
cases, and there should be marks indicating the N. A. of the condenser 
utilized with different openings. Finally the condenser should be 
supplied with central stops for dark-ground illumination (§92) and 
with blue and neutral tint glasses to soften the glare when artificial 
light is used (§ 89, 93). 

Condensers or Illuminators fall into two great groups, the 
Achromatic, giving a large aplanatic cone, and Non-achromatic, 
giving much light, but a relatively small aplanatic cone of light. 

§ 80. Achromatic Condenser.— It is still believed by all expert 
microscopists that the contention of Brewster was right, and the con- 
denser to give the greatest aid in elucidating microscopic structure 
must approach in excellence the best objectives. That is, it should be 
as free as possible from spherical and chromatic aberration, and there- 
fore would transmit to the object a very large aplanatic cone of light. 
Such condensers are especially recommended for photo-micrography by 
all, and those who believe in getting the best possible image in every 
case are equally strenuous that achromatic condensers should be used 
for all work. Unfortunately good condensers like good objectives are 
expensive, and student microscopes as well as many others are usually 
supplied with the non-achromatic condensers or with none. 

Many excellent achromatic condensers have been made, but the 
most perfect of all seems to be the apochromatic of Powell and Lealand 
(Carpenter-Dallinger, p. 302). To attain the best that was possible 
many workers have adopted the plan of using objectives as condensers. 
A special substage fitting is provided with the proper screw and the 
objective is put into position, the front lens being next the object. As 
will be seen below (§ 83-84), the full aperture of an objective can 
rarely be used, and for histological preparations perhaps never, so that 
an objective of greater equivalent focus, i. e., lower power is used for 
the condenser than the one on the microscope. It is much more con- 
venient, however, to have a special condenser with iris diaphragm or 
special diaphragms so that one may use any aperture at will, and thus 
satisfy the conditions necessary for lighting different objects for the 
same objective and for lighting with objectives of different apertures. 
An excellent condenser of this form has been produced by Zeiss (Fig. 
41). It has a total numerical aperture of 1.00, and an aplanatic aper- 
ture of 0.65. 



CH. II] 



LIGHTING AND FOCUSING 



43 



Fig. 41. Zeiss' 1 Achromatic Conden- 
ser, c. s. c. s. Centering screws for 
changing the position of the condenser 
and making its axis continuous with 
that of the microscope. A segment of 
the condenser is cut aioay to show the 
combinations of lenses. For very low 
powers the upper lens is sometimes 
screwed off. There is an iris dia- 
phragm between the middle and lower 
combinations. [Zeiss' 1 Catalog). 




§ Si. Centering the Condenser. — To get the best possible 
illumination for bringing out in the clearest manner the minute details 
of a microscopic object two conditions are necessary, viz.: The princi- 
pal optic axis of the condenser must be continuous with that of the 
microscope (see frontispiece) and the object must be in the focus of the 
condenser, i. <?., at the apex of the cone of light given by the condenser. 

The centering is most conveniently accomplished as follows 
although daylight may be used with almost equal facility. A very 
small diaphragm is put below the condenser. (If the Zeiss achromatic 
condenser is used, the diaphragm of the Abbe illuminator serves for 
this. If there is no pin-hole diaphragm one can be made of stiff, 
black paper. Care must be taken, however, to make the opening ex- 
actly central. This is best accomplished by putting the paper disc over 
the iris or metal diaphragms and then making the hole in the center of 
the small circle uncovered by the metal diaphragm. For the hole a fine 
needle is best).. light well and lower the objective so that it is at 
about its working-distance from the top of the condenser. If now the 
condenser is lowered or racked away from the objective the image of 
the diaphragm will appear. If the opening is not central it should be 
made so by using the centering screws of the condenser. 

A better plan than to lower the condenser to focus the image of the 
diaphragm, is to raise the body of the microscope slowly with the coarse 
adjustment. It is almost impossible to make apparatus so accurate that 
two parts like the body of the microscope and the substage, each work- 
ing on different sliding surfaces, shall continue in exactly the same 
plane. So one will find that if the condenser be accurately centered 
with the condenser lowered, and then the condenser be racked up close 
to the stage and the image of the diaphragm opening brought again 
into focus by racking up the body of the microscope, it will not be 





44 LIGHTING AND FOCUSING [CH. II 

accurately centered in most cases. For this reason it is advised that 
the condenser be left in position close to the stage and the tube of the 
microscope be used to focus the diaphragm exactly as in ordinary 
work. 

Fig. 42 . Shows that the optic axis of 
the condenser does not coincide with that 
of the microscope. (D). Image of the 
diaphragm of the condenser shown at 
one side of the field of view . 

Fig. 43. Shows the image of the 
diaphragm (D) in the center of the field 
of the microscope, and thus the coin- 
Exc c cidence of the axis of the condenser with 

that of the microscope. 

Fig. 42 Fig. 43 

§ 82. Centering the Image of the Source of Illumination. — 
For the best results it is not only necessary that the condenser be pro- 
perl} 7 centered, but that the object to be studied should be in the image 
of the source of illumination and that this should also be centered 
(Figs. 44, 45). After the condenser itself is centered the iris diaphragm 
is opened to its full extent or the diaphragm carrier turned wholly 
aside. A transparent specimen like the fly's wing is put under the 
microscope and focused. The condenser is then turned up and down 
until the image of the flame is apparently on the specimen. If this 
cannot be accomplished the relative position of the lamp and condenser 
is not correct and should be so changed that the image of the edge of 
the flame is sharply defined. This image must also be centered. This 
is easily accomplished by manipulation of the mirror 'or, if a lamp is 
used, by changing the position of the lamp or of the bull's eye 

(Fig- 53)- 

§83. Proper Numerical Aperture of the Condenser. — As 

stated above, the aperture - of the condenser should have a range by 
means of properly selected diaphragms to meet the requirements of all 
objectives from the lowest to those of the highest aperture. It is 
found in practice that for diatoms, etc., the best images are obtained 
when the object is lighted with a cone which fills about three- fourths 
of the diameter of the back lens of the objective with light but for 
histological and other preparations of lower refractive power only one- 
half or one-third the aperture often gives the most satisfactory images 
(§34)- 



CH. II] 



LIGHTING AND FOCUSING 



45 



Fig. 44. Shows the image of the 
flame {Ft.} in the center (C) of the 
field of the microscope and illuminat- 
ing the object. 

Fig. 45. Shows the image of the 
flame [Ft. ) at one side of the centre 
(Exc.) and not properly illuminating 
the object. 





Fig. 44. 



Fig. 45- 



To determine this in any case focus upon some very transparent 
object, takeout the ocular, look down the tube at the back lens. If less 
than three-fourths of the back lens is lighted, increase the opening in 
the diaphragm— if more than three-fourths diminish it. For some 
objects it is advantageous to use less than three-fourths of the aper- 
ture. Experience will teach the best lighting for special cases. 



Obj 



Obj 






III u 



Ilium 



Fig. 46. 



Fig. 47. 



Figs. 46-47. Figures showing the depeyidence of the objective upon the ilium 
inciting cone of the condenser {Nelson). 

Fig. 46(A). The illuminating cone from the condenser {Ilium). This is 
seen to be just sufficient to fill the objective ( Obj). 

(B). The back lens of the objective entirely filled with light, showing that the 
numerical aperture of the illuminator is equal to that of the objective. 

Fig. 47 [A). In this figure the illuminating conefrome the condenser (Ilium.) 
is seen to be insufficient to fill the objective ( Obj). 

(B). The back lens of the objective only partly filled with light, due to the 
restricted aperture of the illuminator. 

§ 84. Aperture of the Illuminating Cone and the Field. — It 

is to be remarked that with a very small source of light the entire aper- 
ture of the objective may be filled if a proper illuminator or condenser 
is used. The aperture depends on the diaphragm used with the con- 
denser. And the size of the diaphragm must be directly as the aper- 
ture of the objective. That is, it is just the reverse of the rule for 
diaphragms where no condenser is used (§ 67) ; for there the diaphragm 



46 LIGHTING AND FOCUSING [CH. II 

is made large for low powers, and consequently low apertures, while 
with the condenser the diaphragm is made small for low and large for 
high powers as the aperture is greater in the high powers of a given 
series of objectives. It is very instructive to demonstrate this by using 
a 16 mm. objective and opening the diaphragm of the condenser till the 
back lens is just filled with light. Then if one uses a 3 or 4 mm. ob- 
jective it will be seen that the back lens of the higher objective is only 
partty filled with light and to fill it the diaphragm must be much more 
widely opened. 

With a condenser, then, the diaphragm has simply to regulate the 
aperture of the illuminating cone, and has nothing to do with lighting 
a large or a small field. 

With the condenser there are two conditions that must be fulfilled, 
— the proper aperture must be used, and that is determined by the dia- 
phragm, and secondly the whole field must be lighted. The latter is 
accomplished by using a larger source of light, as the face instead of 
the edge of a lamp flame, or by lowering or raising the condenser so 
that the object is not in the focus of the condenser, but above or below 
it, and therefore lighted by a converging or diverging beam where the 
light is spread over a greater area (Figs. 48-51, § 88). 

§ 85. Non- Achromatic Condenser. — Of the non-achromatic 
condensers or illuminators, the Abbe condenser or illuminator is the 
one most generally used. From its cheapness it is also much more com- 
monly used than the achromatic condenser. It consists of two or three 
very large lenses and transmits a cone of light of 1.20 N. A. to 1.40 N. 
A., but the aberrations, both spherical and chromatic, are very great in 
both forms. Indeed, so great are they that in the best form of three 
lenses with an illuminating cone of 1.40 N. A., the aplanatic cone 
transmitted is only 0.5, and it is the aplanatic cone which is of real use 
in microscopic illumination where details are to be studied. There is 
no doubt, however, that the results obtained with a non-achromatic 
condenser like the Abbe are much more satisfactory than with no con- 
denser. The highest results cannot be attained with it, however. 
( Carpenter- Dallinger, p. 309). 

§ 86. Arrangement of the Condenser. — The proper position of 
the illuminator for high objectives is one in which the beam of light 
traversing it is brought to a focus on the object. If parallel rays are 
reflected from the plane mirror to it, they will be focused only a few 
millimeters above the upper lens of the condenser ; consequently the 
illuminator should be about on the level of the top of the stage and 



CH. IT] LIGHTING AND FOCUSING 47 

therefore almost in contact with the lower surface of the slide. For 
some purposes when it is desirable to avoid the loss of light by reflec- 
tion or refraction, a drop of water or homogeneous immersion fluid is 
put between the slide and condenser, forming the so-called immersion 
illuminator. This is necessary only with objectives of high power 
and large aperture or for dark-ground illumination. 

§ 87. Centering the Condenser. — The illuminator should be 
centered to the optic axis of the microscope, that is the optic axis of 
the condenser and of the microscope should coincide. Unfortunately 
there is extreme difficult}' in determining when the Abbe illuminator is 
centered. Centering is approximated as follows : Put a pin-hole dia- 
phragm — that is a diaphragm with a small central hole — over the end 
of the condenser (Fig. 52), the central opening should appear to be in 
the middle of the field of the microscope. If it does not the condenser 
should be moved from side to side by loosening the centering screws 
until it is in the center of the field. In case no pin-hole diaphragm 
accompanies the condenser, one may put a very small drop of ink, as 
from a pen-point, on the center of the upper lens and look at it with 
the microscope to see if it is in the center of the field. If it is not, 
the condenser should be adjusted until it is. When the condenser is 
centered as nearly as possible remove the pin-hole diaphragm or the 
spot of ink. The microscope and illuminator axes may not be entirely 
coincident even when the center of the upper lens appears in the cen- 
ter of the field, as there may be some lateral tilting of the condenser, but 
the above is the best the ordinary worker can do, and unless the 
mechanical arrangements of the illuminator are very deficient, it will 
be very nearly centered. 

It is to be hoped that the opticians will devise some kind of 
mounting for this the most commonly used condenser whereby it may 
be centered as described for the achromatic condenser instead of by the 
crude methods described above. If the condenser mounting regularly 
possessed centering screws as in the microscope of Watson & Sons and 
there were a centering diaphragm in the proper position so that its im- 
age could be projected into the field of view, the operation would be 
very simple. If, further, the condensers of Powell and Lealand were 
selected as models the condensers need not be so bulky, and would still 
retain all their efficiency. 

Fortunately the Royal Microscopical Society of London which has 
done so much toward standardizing microscopical apparatus has recently 
proposed as a standard size for the substage fitting for the condenser of 
1.527 in. = 38.786 mm. (see § 46). 



4« LIGHTING AND FOCUSING [CH. II 

§ 88. Mirror and Light for the Abbe Condenser. — It is best to 
use light with parallel rays. The rays of daylight are practically par- 
allel ; it is best therelore to employ the plane mirror for all but the 
lowest powers. If low powers are used the whole field might not be 
illuminated with the plane mirror when the condenser is close to the 
object ; furthermore, the image of the window frame, objects outside 
the building, as trees, etc., would appear with unpleasant distinctness 
in the field of the microscope. To overcome these defects one can 
lower the condenser and thus light the object with a diverging cone of 
light, or use the concave mirror and attain the same end when the con- 
denser is close to the object (Fig. 48). 

§ 89. Artificial Light. — If one uses lamp light, it is recommend- 
ed that a large bull's eye be placed in such a position between the 
light and the mirror that parallel rays fall upon the mirror or in some 
cases an image of the lamp flame. If one does not have a bull's eye 
the concave mirror may be used to render the rays less divergent. It 
may be necessary to lower the illuminator somewhat in order to illum- 
inate the object in its focus. 

ABBE CONDENSER : EXPERIMENTS 

§ 90. Abbe Condenser, Axial and Oblique Light. — Use a dia- 
phragm a little larger than the front lens of the 3 mm. (j/£ in.) objec- 
tive, have the illuminator on the level, or nearly on the level of the 
upper surface of the stage, and use the plane mirror. Be sure that 
the diaphragm carrier is in the notch indicating that it is central in 
position. Use the Pleurosigma as object. Study carefully the appear- 
ance of the diatom with this central light, then make the diaphragm 
eccentric so as to light with oblique light (§78). The differences in 
appearance will probably be even more striking than with the mirror 
alone. 

§91. Lateral Swaying of the Image. — Frequently in study- 
ing an object, especially with a high power, it will appear to sway 
from side to side in focusing up or down. A glass stage micrometer or 
fly's wing is an excellent object. Make the light central or axial and 
focus up and down and notice that the lines simply disappear or grow 
dim. Now make the light oblique, either by making the diaphragm 
opening eccentric or if simply a mirror is used, by swinging the mirror 
sidewise. On focusing up and down, the lines will sway from side to 
side. What is the direction of apparent movement in focusing down 



CH. IT] 



LIGHTING AND FOCUSING 



49 



with reference to the illuminating ray ? What in focusing up ? If one 
understands the experiment it may sometimes save a great deal of con- 
fusion. (See under testing the microscope for swaying with central 
light § 119.) 

§ 92. Dark-Ground Illumination. — When an object is lighted 
with rays of a greater obliquity than can get into the front lens of the 
objective, the field will appear dark (Fig. 51). If now the object is 




Figs. 48-51. Sectional views of the Abbe Illuminator of 1 .20 N. A. showing 
various methods of illumination (§ qo). Fig. 48, axial light with parallel rays. 
Fig. 49, oblique light. Fig. 50, axial light with converging beam. Fig. 51, dark- 
ground illumination with a central stop diaphragm. 

Axis. The optic axis of the illuminator and of the microscope. The illumi- 
nator is centered, that is its optic axis is a prolongation of the optic axis of the 
microscope. 

S. Axis. Secondary axis. In oblique light the central ray passes along a 
secondary axis of the illuminator, and is therefore oblique to the principal axis. 

D. D. Diaphragms. These are placed in sectional and in face views. The 
diaphragm is placed between the mirror and the illuminator. In Fig. 49 the open- 
ing is eccentric for oblique light, and in Fig. 5/ the opening is a narrow ring, the 
central part being stopped out, thus giving rise to dark-ground illumination ($ 92). 

Obj. Obj. The front of the objective. 

composed of fine particles, or is semi-transparent, it will refract or 
reflect the light which meets it, in such a way that a part of the very 



5° 



LIGHTING AND FOCUSING 



\CH, II 




oblique rays will pass into the objective, hence as light reaches the 
objective onty from the object, all the surrounding field will be dark 
and the object will appear "like a self-luminous one on a dark back- 

Fig. 52. An Abbe Condenser in its mounting 
{The Bausch & Lomb Optical Company). 

ground. This form of illumination is most 
successful with low powers. It is well to 
make the illuminator immersion for this 
experiment, (see § 105). 

(A) With the Mirror— Remove all the 
diaphragms so that very oblique light may 
be used, employ a stage micrometer in 
which the lines have been filled with graph- 
ite, use a 16 mm. (7/3 in.) objective, and when the light is sufficiently 
oblique the lines will appear something like streaks of silver on a 
black back-ground. A specimen like that described below in (B) may 
also be used. 

(B) With the Abbe Condeiiser. — Have the illuminator so that the 
light is focused on the object (see § 86) and use a diaphragm with 
the annular opening (Fig. 51); employ the same objective as in 
(A). For object place a drop of 10 % solution of salicylic acid in 95 % 
alcohol on the middle of a slide ; it will crystallize. The crystals will 
appear brilliantly lighted on a dark back-ground. Put in an ordinary 
diaphragm and make the light oblique by making the diaphragm 
eccentric. The same specimen may also be tried with a mirror and 
oblique light. In order to appreciate the difference between this dark- 
ground and ordinary transmitted-light illumination, use an ordinary 
diaphragm and observe the crystals. 

A very striking and instructive experiment may be made by add- 
ing a very small drop of the solution to the dried preparation, putting 
it under the microscope quickly, lighting for dark-ground illumination 
and then watching the crystallization. 



ARTIFICIAL ILLUMINATION 

§ 93. For evening work and for regions where daylight is not 
sufficiently brilliant, artificial illumination must be employed. Fur- 
thermore, for the most critical investigation of bodies with fine mark- 
ings like diatoms, artificial light has been found superior to daylight. 

A petroleum (kerosene) lamp with flat wick gives a satisfactory 
light. It is recommended that instead of the ordinary glass chimney, 



CH. //] 



LIGHTING AND FOCUSING 



51 



one made of metal with a slit-opening covered with an oblong cover- 
glass is more satisfactory, as the source of light is more restricted. 
Very excellent results may be obtained, however, with the ordinary 
bed-room lamp furnished with the usual glass chimney. 

The new acetylene light promises to be excellent for micro- 
scopic observation and for photo-micrography. (See under photo- 
micrography.) 




Fig. 53. 1. Lamp with slit-opening in metal chimney, 
separate stand. 3. Screen showing image of flame. 



2. Bulls eye on 



Whenever possible the edge of the flame is turned toward the 
microscope, the advantage of this arrangement is the great brilliancy, 
due to the greater thickness of the flame in this direction. 

§ 94. Mutual Arrangement of Lamp, Bull's Eye and Micro- 
scope. — To fulfill the conditions given above, namely, that the object 
be illuminated \>y the image of the source of illumination the lamp 
must be in such a position that the condenser projects a sharp image 
of the flame upon the object (Fig. 53), and only by trial can this posi- 
tion be determined. In some cases it is found advantageous to discard 
the mirror and allow the light from the bull's eye to pass directly into 
the condenser. This method is especially excellent in photomicro- 
graphy (see Ch. VIII). 

§ 95. Illuminating the Entire Field. — With low objectives 
and large objects, the entire object might not be illuminated if the 
above method were strictly followed ; in this case turn the lamp so 
that the flame is oblique, or if that is not sufficient, continue to turn 
the lamp until the full width of the flame is used. If necessary the 



52 



LIGHTING AND FOCUSING 



[CH. II 



condenser may be lowered, and the concave mirror used. (See 
also § 84.) 

REFRACTION AND COLOR IMAGES 

I 96. Refraction Images are those mostly seen in studying microscopic 
objects. They are the appearances produced by the refraction of the light on 
entering and on leaving an object. They therefore depend (a) on the form of the 
object, (b) on the relative refractive powers of object and mounting medium. 
With such images the diaphragm should not be too large (see I S3). 

If the color and refractive index of the object were exactly like the mount- 
ing medium it could not be seen. In most cases both refractive index and color 
differ somewhat, there is then a combination of color and refraction images which 
is a great advantage. This combination is generally taken advantage of in histol- 
ogy. The air bubble in \ 77 is an example of a purely refractive image. 




Figs. 54-56. Diagrams illustrating refraction in different media and at plane 
and curved surfaces. In each case the denser medium is represented by line shad- 
ing and the perpendicular or normal to the refracting surface is represented by the 
dotted line N-N' , the refracted ray by the bent line A C. 

\ 97. Refraction. — Lying at the basis of microscopical optics is refraction, which 
is illustrated by the above figures. It means that light passing from one medium 
to another is bent in its course. Thus in Fig. 54 light passing from air into water 
does not continue in a straight line but is bent toward the normal N-N 7 , the 
bending taking place at the point of contact of the air and water ; that is, the ray 
of light A B entering the water at B is bent out of its course, extending to C 
instead of C / . 

Conversely, if the ray of light is passing from water into air, on reaching the 
air it is bent from the normal, the ray C B passing to A and not in a straight line 
to C /r . By comparing Figs. 55, 56 in which the denser medium is crown glass in- 
stead of water, the bending of the rays is seen to be greater as crown glass is 
denser than water. 

It has been found by physicists that there is a constant relation between the 
angle taken by the ray in the rarer medium and that taken by the ray in the 
denser medium. The relationship is expressed thus : Sine of the angle of inci- 



CH. II] LIGHTING AND FOCUSING 53 

dence divided by the sine of the angle of refraction equals the index of refraction. 

In the figures, —. — _ „ „ T , = index of refraction. Worked out completely in 
s Sm CBN' 

Fig. 54, A B N=4o°, CB N'= 28° 54' and _? in *° ° - =-^^^ = 1.33, i.e., 
°*' H ' °^ Sin 28 54' 0.48327 °° 

the index of refraction from air to water is 1.33. (See \ 33.) In Figs. 55-56, 

illustrating refraction in crown glass, the angles being given, the problem is easily 

solved as just illustrated. (For table of natural sines see third page of cover ; for 

interpolation, \ 32). 

I 98. Absolute Index of Refraction. — This is the index of refraction obtained 
when the incident ray passes from a vacuum into a given medium. As the index 
of the vacuum is taken as unity, the absolute index of any substance is always 
greater than unity. For many purposes, as for the object of this book, 
air is treated as if it were a vacuum, and its index is called unity, but in reality 
the index of refraction of air is about 3 ten-thousandths greater than unity. 
Whenever the refractive index of a substance is given, the absolute index is 
meant unless otherwise stated. For example, when the index of refraction of 
water is said to be 1.33, and of crown glass 1.52, etc., these figures represent the 
absolute index, and the incident ray is supposed to be in a vacuum. 

§ 99. Relative Index of Refraction. — This is the index of refraction between 
two contiguous media, as for example between glass and diamond, water and 
glass, etc. It is obtained by dividing the absolute index of refraction of the sub- 
stance containing the refracted ray, by the absolute index of the substance trans- 
mitting the incident ray. For example, the relative index from water to glass is 
1.52 divided by 1.33. If the light passed from glass to water it would be, 1.33 
divided by 1.52. 

By a study of the figures showing refraction, it will be seen that the greater the 

refraction the less the angle and consequently the less the sine of the angle, and as 

the refraction between two media is the ratio of the sines of the angles of incidence 

/ sin i \ 

and refraction ( — ) , it will be seen that whenever the sine of the angle of refrac- 

\sm r / 

tion is increased by being in a less refractive medium, the index of refraction will 

show a corresponding decrease and vice versa. That is the ratio of the sines of the 

angles of incidence and refraction of any two contiguous substances is inversely as 

the refractive indices of those substances. The formula is : 

/ Sine of angle of incident ray \ / Index of refraction of refracting medium \ 

V Sine of angle of refracted ray / \ Index of refraction of incident medium / 

Abbreviated ( -. ) = ( ; — z . ) • By means of this general formula one can 

\sin rj \ index z/ 

solve any problem in refraction whenever three factors of the problem are known. 

The universality of the law may be illustrated by the following cases : 

(A) Light incident in a vacuum or in air, and entering some denser medium, 

as water, glass, diamond, etc. 

/ Sine of angle made by the ray in air \ / Index of ref .of denser med. \ 

V. Sine of angle made by ray in denser medium / \ Index of ref. of air ( 1) / 

/ gin i \ / 1 o \ 

If the dense substance were glass I I I 1 • If the two media were 

\sin r J — \ 1 / 



54 LIGHTING AND FOCUSING [CH. II 

water and glass, the incident light being in water the formula would be : 

( ) = ( — : — I • If the incident ray were in glass and the refracted ray in 

\sin rj V i-33 / 

water : ( — ) =• ( ) • And similarly for any two media ; and as stated 

\sin rj \ 1.52 / 

above if any tbree of the factors are given the fourth may be readily found. 

\ 100. Critical Angle and Total Reflection. — In order to understand the Wol- 
laston camera lucida (Ch. IV) and other totally reflecting apparatus, it is necessary 
briefly to consider the critical angle. 

The critical angle is the greatest angle that a ray of light in the denser of two 
contiguous media can make with the normal and still emerge into the less refrac- 
tive medium. On emerging it will form an angle of 90 with the normal, and if 
the substances are liquids, the refracted ray will be parallel with the surface of the 
denser medium. 

Total Reflection. — In case the incident ray in the denser medium is at an angle 
with the normal greater than the critical angle, it will be totally reflected at the 
surface of the denser medium, that surface acting as a perfect mirror. By consult- 
ing the figures it will be seen that there is no such thing as a critical angle and 
total reflection in the rarer of two contiguous media. 

To find the critical angle in the denser of two contiguous media : — 

Make the angle of refraction (z. e. , the angle in the rarer of the two media) 

• / sin 1 \ / index r \ 

qo° and solve the general equation : ( — I = I -■ — ; r ) . Let the two sub- 

\sin r J \ index 1 / 

stances be water and air, then the sine of r (90 ) is 1, the index of air is 1, that of 
water 1.33, whence ( ) = ( ) or S1 V z = 75 I_ K This is the sine of 

48° + , and whenever the ray in the water is at an angle of more than 4S it will 
not emerge into the air, but be totally reflected back into the water. 

The case of a ray passing from crown glass into the water : 

/ sin i \ /index water ( 1.33) \ / sin i \ / 1.33 \ 

\sin r (sin 90°= 1) / ~~ \ index glass (1.52) / \ 1 / V1.52/ 
whence sin i =.875 sine of critical angle in glass covered with water. The 
corresponding angle is approximately 6i°. 

\ 101. Color Images. — These are images of objects which are strongly col- 
ored and lighted with so wide an aperture that the refraction images are drowned 
in the light. Such images are obtained by removing the diaphragm or by using a 
larger opening. This method of illumination is especially applicable to the study 
of deeply stained bacteria. (See below. § 108). 

ADJUSTABLE WATER AND HOMOGENEOUS OBJECTIVES 
EXPERIMENTS 

I 102. Adjustment for Objectives. — As stated above (§ 24), the aberration 
produced by the cover-glass (Fig. 57), is compensated for by giving the combina- 
tions in the objective a different relative position than they would have if the 
objective were to be used on uncovered objects. Although this relative position 
cannot be changed in unadjustable objectives, one can secure the best results of 



CH. II] 



LIGHTING AND FOCUSING 



55 




which the objective is capable by selecting covers of the thickness for which the 
objective was corrected. (See table p. 14.) Adjustment maybe made also by 
increasing- the tube-length for co'vers thinner than the standard and by shortening 
the tube-length for covers thicker than the standard (Fig. 5S). 

Fig. 57. Effect of the cover-glass 
on the rays from the object to the 
objective {Ross). 

Axis. The projection of the optic 
axis of the microscope. 

F. Focal or axial point of the 
objective. 

F' and F" '. Points on the axis 
where rays 2 and 3 appear to originate 
if traced backward after emerging 
from the upper side of the cover-glass. 

In learning to adjust objectives, it is best for the student to choose some 
object whose structure is well agreed upon , and then to practice lighting it, shad- 
ing the stage and adjusting the objective, until the proper appearance is obtained. 
The adjustment is made by turning a ring or collar which acts on a screw and 
increases or diminishes the distance between the systems of lenses, usually the 
front and the back systems (Fig. 40). 

§103. General Directions. — (A) The thinner the cover-glass, the 
further must the system of lenses be separated, i. e. , the adjusting collar 
is turned nearer the zero or the mark "uncovered," and conversely; (B) 
the thicker the cover-glass the closer together are the systems brought 
by turning the adjusting collar from the zero mark. This also increases 
the magnification of the objective (Ch. IV). 

The following specific directions for making the cover-glass adjust- 
ment are given by Mr. Wenham (Carpenter, 7th Ed., p. 166). "Select 
any dark speck or opaque portion of the object, and bring the outline 
into perfect focus ; then lay the finger on the milled-head of the fine 
motion, and move it briskly backwards and forwards in both directions 
from the first position. Observe the expansion of the dark outline of 
the object, both when within and when without the focus. If the 
greater expansion or coma is when the object is without the focus, or 
farthest from the objective [z. e., in focusing up], the lenses must be 
placed further asunder, or toward the mark uncovered [the adjusting 
collar is turned toward the zero mark as the cover-glass is too thin for 
the present adjustment]. If the greater expansion is when the object 
is within the focus, or nearest the objective [z. <?. , in focusing down], 
the lenses must be brought closer together, or toward the mark covered 
[z. e., the adjusting collar should be turned away from the zero mark, 



56 LIGHTING AND FOCUSING \CH. II 

the cover-glass being too thick for the present adjustment]." In most 
objectives the collar is graduated arbitrarily , the zero (O) ?nark represent- 
ing the position for tincovered objects. Other objectives have the collar 
graduated to correspotid to the various thicknesses of cover-glasses for which 
the objective may be adjusted. This seems to be an admirable plan ; then 
if one knows the thickfiess of the cover-glass on the preparation (Ch. VIII) 
the adjusting collar may be set at a corresponding mark, and o?ie will feel 
confident that the adjustment will be approximately correct. It is then 
o?ily necessary for the observer to make the slight adjustment to compensate 
for the mounting medium or any variation from the standard length of the 
tube of the microscope. In adjusting for variations of the length of 
the tube from the standard it should be remembered that : (A) If the 
tube of the microscope is longer than the standard for which the ob- 
jective was corrected, the effect is approximately the same as thicken- 
ing the cover-glass, and therefore the system of the objective must be 
brought closer together, i. e., the adjusting collar must be turned away 
from the zero mark. (B) If the tube is shorter than the standard for 
which the objective is corrected, the effect is approximately the same as 
diminishing the thickness of the cover-glass, and the systems must 
therefore be separated (Fig. 40). 

In using the tube-length for cover correction Shorten the tube for 
too thick covers and Lengthen the tube for too thin covers. 

Furthermore, whatever the interpretation by different opticians of 
what should be included in "tube-length," and the exact length in mil- 
limeters, its importance is very great ; for each objective gives the most 
perfect image of which it is capable with the "tube-length" for which 
it is corrected, and the more perfect the objective the greater the ill- 
effects on the image of varying the "tube-length" from this standard. 
The plan of designating exactly what is meant by "tube-length," and 
engraving on each objective the "tube-length" for which it is corrected, 
is to be commended, for it is manifestly difficult for each worker with 
the microscope to find out for himself for what "tube-length" each of 
his objectives was corrected. (See Ch. X). 

§ 104. Water Immersion Objectives. — Put a water immersion 
objective in position (§ 47) and the fly's wing for object under the 
microscope. Place a drop of distilled water on the cover-glass, and 
with the coarse adjustment lower the tube till the objective dips into 
the water, then light the field well and turn the fine adjustment one 
way and another till the image is clear. Water immersions are exceed- 
ingly convenient in studying the circulation of the blood, and for 



CH. II] 



LIGHTING AND FOCUSING 



57 



mam* other purposes where aqueous liquids are liable to get on the 
cover-glass. If the objective is adjustable, follow the directions given 
in § 103. 




Objeetive 



Object-b 
Object-a 



Fig. 58. Figure to show that in lengthening the tube of the microscope the ob- 
ject must be brought nearer the principal focus or center of the lens. It will be seen 
by consulting the figure that in shortening the tube of the microscope the object 
must be removed farther from the center of the lens. By consulting the figure 
showing the effect of the cover-glass {Fig. 57) it will be seen that the effect of the 
cover-glass is to bring the object nearer the objective, and the thicker the cover the 
nearer is the object brought to the objective. As shortening the tube serves to remove 
the object, it neutralizes the effect of the thick cover, and if the cover is so thin that 
it does not elevate the object enough for the corrections of the objective, then an in- 
crease in the tube-length will correct the defect. 



5§ LIGHTING AND FOCUSING \CH. II 

When one is through using a water immersion objective, remove it 
from the microscope and with some lens paper wipe all the water from 
the front lens. Unless this is done dust collects and sooner or later 
the front lens will be clouded. It is better to use distilled water to 
avoid the gritty substances that are liable to be present in natural 
waters, as these gritty particles might scratch the front lens. 

HOMOGENEOUS IMMERSION OBJECTIVES : EXPERIMENTS 

§ 105. As stated above, these are objectives in which a liquid of 
the same refractive index as the front lens of the objective is placed 
between the front lens and the cover-glass. 

§ 106. Tester for Homogeneous Liquid. — In order that full 
advantage be derived from the homogeneous immersion principle, the 
liquid employed must be truly homogeneous. To be sure that such is 
the case, one may use a tester like that constructed by the Gundlach 
Optical Co. , then if the liquid is too dense it may be properly diluted 
and vice versa. For the cedar oil immersion liquid, the density may 
be diminished by the addition of pure cedar wood oil. The density 
may be increased by allowing it to thicken by evaporation. (See H. 
L. Smith, Proc. Amer. Soc. Micr., 1885, p. 83, and Ch. X). 

§107. Refraction Images. — Put a 2 mm. (y^th in.) homogeneous 
immersion objective in position, employ an illuminator. Use some 
histological specimen like a muscular fiber as object, make the dia- 
phragm opening about 3 mm. in diameter, add a drop of the homo- 
geneous immersion liquid and focus as directed in § 74. The object 
will be clearly seen in all details by the unequal refraction of the light 
traversing it. The difference in color between it and the surrounding 
medium will also increase the sharpness of the outline. If an air bub- 
ble preparation (§ 77) were used, one would get pure, refraction 
images. 

§ 108. Color Images. — Use some stained bacteria as Bacillus 
tuberculosis for object. Put a drop of the immersion liquid on the 
cover-glass or the front lens of the homogeneous objective. Remove 
the diaphragms from the illuminator or in case the iris diaphragm is 
used, open to its greatest extent. Focus the objective down so that 
the immersion fluid is in contact with both the front lens and the cover- 
glass, then with the fine adjustment get the bacteria in focus. They 
will stand out as clearly defined colored objects on a bright field. 



CH. II} LIGHTING AND FOCUSING 59 

Fig. 59. Screen for shading the microscope and 
the face of the observer. This is very readily con- 
structed as shown in the figure by supporting a wire 
in a disc of lead, iron, or heavy wood. The screen is 
then completed by hanging over the bent'wire, cloth or 
manilla paper 30 x 40 cm. The lower edge of the 
screen should be a little below the stage of the micro- 
scope and the upper edge high enough to screen the- 
eyes of the observer. 

§ 109. Shading the Object.— To get the 
clearest image of an object no light should 
reach the eye except from the object. A hand- 
kerchief or a dark cloth wound around the 

objective will serve the purpose. Often the proper effect may be ob- 
tained by simply shading the top of the stage with the hand or with a 
piece of bristol board. Unless one has a very favorable light the shading 
of the object is of the greatest advantage, especially with homogeneous 
immersion objectives. The screen (Fig. 59) is the most satisfactory 
means for this purpose, as the entire microscope above the illuminating 
apparatus is shaded. 

§ no. Cleaning Homogeneous Objectives. — After one is 
through with a homogeneous objective, it should be carefully cleaned as 
follows : Wipe off the homogeneous liquid with a piece of the lens 
paper (§ 114), then if the fluid is cedar oil, wet one corner of a fresh 
piece in benzin or chloroform and wipe the front lens with it. Imme- 
diately afterward wipe with a dry part of the paper. The cover-glass 
of the preparation can be cleaned in the same way. If the homogen- 
eous liquid is a glycerin mixture proceed as above, but use water to 
remove the last traces of glycerin. 

CARE OF THE MICROSCOPE 

§111. The microscope should be handled carefully and kept per- 
fectly clean. The oculars and objectives should never be allowed to 
fall. 

When not in use keep it in a place as free as possible from dust. 

All parts of the microscope should be kept free from liquids, 
especially from acids, alkalies, alcohol, benzin, turpentine and 
chloroform. 

§ 112. Care of the Mechanical Parts. — To clean the mechan- 
ical parts put a small quantity of some fine oil (olive oil or liquid vas- 



60 LIGHTING AND FOCUSING [CH. II 

elin and benzin, equal parts), on a piece of chamois leather or on the 
lens paper, and rub the parts well, then with a clean dry piece of the 
chamois or paper wipe off most of the oil. If the mechanical parts 
are kept clean in this way a lubricator is rarely needed. Where op- 
posed brass surfaces "cut," i. e., when from the introduction of some 
gritty material, minute grooves are worn in the opposing surfaces, giv- 
ing a harsh movement, the opposing parts should be separated, care- 
fully cleaned as described above and any ridges or prominences scraped 
down with a knife. Where the tendency to "cut" is marked, a very 
slight application of equal parts of beeswax and tallow, well melted 
together, serves a good purpose. 

In cleaning lacquered parts, benzin alone answers well, but it 
should be quickly wiped off with a clean piece of the lens paper. Do 
not use alcohol as it dissolves the lacquer. 

§ 113. Care of the Optical Parts. — These must be kept scrupu- 
lously clean in order that the best results may be obtained. 

Glass surfaces should never be touched with the fingers, for that 
will soil them. 

The glass of which the lenses are made is quite soft, consequently 
it is necessary that only soft, clean cloth or paper be used in wiping 
them. 

Whenever an objective is left in position on a microscope, or when 
several are attached by means of a revolving nose-piece, an ocular 
should be left in the upper end of the tube to prevent dust from falling 
down upon the back lens of the objective. 

§114. Lens Paper. — The so-called Japanese filter paper, which 
from its use with the microscope, I have designated lens paper, has 
been used in the author's laboratory for the last sixteen years for clean- 
ing the lenses of oculars and objectives, and especially for removing 
the fluid used with immersion objectives. Whenever a piece is used 
once it is thrown away. It has proved more satisfactory than cloth or 
chamois, because dust or sand is not present ; and from its bibulous 
character it is very efficient in removing liquid or semi-liquid substances. 

§ 115. Dust may be removed with a camel's hair brush, or by 
wiping with the lens paper. 

Cloudiness may be removed from the glass surfaces by breathing 
on them, then wiping quickly with a soft cloth or the lens paper. 

Cloudiness on the inner surfaces of the ocular lenses may be re- 
moved by unscrewing them and wiping as directed above. A high 
objective should never be taken apart by an inexperienced person. 



CH. II] LIGHTING AND FOCUSING 61 

If the cloudiness cannot be removed as directed above, moisten one 
corner of the cloth or paper with 95 per cent, alcohol, wipe the glass first 
with this, then with' the dry cloth or the paper. 

Water may be removed with soft cloth or the paper. 

Glycerin may be removed with cloth or paper saturated with dis- 
tilled water ; remove the water as above. 

Blood or other albuminous material may be removed while fresh 
with a moist cloth or paper, the same as glycerin. If the material 
has dried on the glass, it may be removed more readily by adding a 
small quantity of ammonia to the water in which the cloth is moistened, 
(water 100 cc, ammonia 1 cc). 

Canada Balsam, damar, paraffin, or any oily substance may be re- 
moved with a cloth or paper wet with chloroform, benzin or xylene. 
The application of these liquids and their removal with a soft dry cloth 
or paper should be as rapid as possible, so that none of the liquid will 
have time to soften the setting of the lenses. 

Shellac Cevient may be removed by the paper or a cloth moistened 
in 95 per cent, alcohol. 

Brunswick Black, Gold Size, and all other substances soluble in 
chloroform, etc., maybe removed as directed for balsam and damar. 

In general, use a solvent of the substance on the glass and wipe it 
off quickly with a fresh piece of the lens paper. 

It frequently happens that the upper surface of the back combina- 
tion of the objective becomes dusty. This may be removed in part by 
a brush, but more satisfactorily by using a piece of the soft paper loosely 
twisted. When most of the dust is removed some of the paper may be 
put over the end of a pine stick (like a match stick) and the glass sur- 
face carefully wiped. 

CARE OF THE EYES 

§ 116. Keep both eyes open, using the eye-screen if necessary (Figs. 
60, 60a); and divide the labor between the two eyes, i. e., use one eye 
for observing the image awhile and then the other. In the begin- 
ning it is not advisable to look into the microscope continuously for 
more than half an hour at a time. One never should work with the 
microscope after the eyes feel fatigued. After one becomes accustomed 
to microscopic observation he can work for several hours with the 
microscope without fatiguing the eyes. This is due to the fact that 
the eyes become inured to labor like the other organs of the body by 
judicious exercise. It is also due to the fact that but very slight ac- 
commodation is required of the eyes, the eyes remaining nearly in a 
condition of rest as for distant objects. The fatigue incident upon 



62 



LIGHTING AND FOCUSING 



[CH. II 



using the microscope at first is due partly at least to the constant effort 
on the part of the observer to remedy the defects of focusing the 
microscope by accommodation of the eyes. This should be avoided 
and the fine adjustment of the microscope used instead of the muscles 
of accommodation. With a microscope of the best qualit3 T , and suita- 
ble light — that is light which is steady and not so bright as to dazzle 
the eyes nor so dim as to strain them in determining details— micro- 
scopic work should improve rather than injure the sight. 

Fig. 60. Double Eye Shade. This is 
readily made by taking some thick bris- 
tol board 7 x 14 centimeters and making 
an oblong opening with rounded ends 
(0-0) and of such a diameter that it goes 
readily over the tube of the microscope. 
This is the?i covered on both sides with 
velveteen and a central slit (5) made in 
the cloth. This admits the tube of the 
microscope and holds the screen in posi- 
tion. It may readily be pulled from side to side and thus serves for either eye, or 
for the use of the eyes alternately . 



7 x 44 


cm. 




,'o 

' s 


\ 


^ > 




Fig. 60 a. Adjusting Eye-Shade. This is prepared like the preceding by cov- 
ering a card about 6 x 12 centimeters with black velveteen. A copper wire about 
? mm.(yi in. ) and of the right length is curved as shown in the figure. Its ends are 
rounded, and finally it is put tinder the cloth and seived carefully all around. The 
card and cloth are then cut as shown. The flexible wire makes it possible to put 
the screen on the tube at any level. 

% 117. Position and Character of the Work-Table. — The 
work-table should be very firm and large (60 x 120 cm.; 24x48 in.), 
so that the necessary apparatus and material for work may not be too 
crowded. The table should also be of the right height to make work 
by it comfortable. An adjustable stool, something like a piano stool is 
convenient, then one ma}- vary the height corresponding to the neces- 



CH. IT] LIGHTING AND FOCUSING 63 

sities of special cases. It is a great advantage to sit facing the window 
if daylight is used, then the hands do not constantly interfere with 
the illumination. To avoid the discomfort of facing the light a screen 
like that shown in Fig. 59 is very useful (see also under lighting, 
§62). 

TESTING THE MICROSCOPE 

I 11S. Testing the Microscope. — To be of real value this must be accom- 
plished by a person with both theoretical and practical knowledge, and also with 
an unprejudiced mind. Such a person is not common, and when found does not 
show over anxiety to pass judgment. Those most ready to offer advice should 
as a rule be avoided, for in most cases they simply "have an ax to grind," and are 
sure to commend only those instruments that conform to the "fad" of the da}'. 
From the writer's experience is seems safe to say that the inexperienced can do no 
better than to state clearly what he wishes to do with a microscope and then trust 
to the judgment of one of the optical companies. The makers of microscopes and 
objectives guard with jealous care the excellence of both the mechanical and 
optical part of their work, and send out only instruments that have been carefully 
tested and found to conform to the standard. This would be done as a matter of 
business prudence on their part, but it is believed by the writer that microscope 
makers are artists first and take an artist's pride in their work, they therefore have 
a stimulus to excellence greater than business prudence alone could give. 

\ 119. Mechanical Parts. — All of the parts should be firm, and not too easily 
shaken. Bearings should work smoothly. The mirror should remain in any 
position in which it is placed. 

Focusing Adjustments. — The coarse or rapid adjustment should be by rack 
and pinion, and work so smoothly that even the highest power can be easily focused 
with it. In no case should it work so easily that the body of the microscope is 
liable to run down and plunge the objective into the object. If any of the above 
defects appear in a microscope that has been used for some time, a person with 
moderate mechanical instinct will be able to tighten the proper screw, etc. 

The Fine Adjustment is more difficult to deal with. Frdm the nature of its 
purpose unless it is approximately perfect, it would be better off the microscope 
entirely. It has been much improved recently. 

It should work smoothly and be so balanced that one cannot tell by the feel- 
ing when using it whether the screw is going up or down. Then there should be 
absolutely no motion except in the direction of the optic axis, otherwise the 
image will appear to sway even with central light. Compare the appearance 
when using the coarse and when using the fine adjustment. There should be no 
swaying of the image with either if the light is central (\ 77). 

\ 120. Testing the Optical Parts. — As stated in the beginning, this can be 
done satisfactorily only by an expert judge. It would be of very great advantage 
to the student if he could have the help of such a person. In no case is a micro- 
scope to be condemned by an inexperienced person. If the beginner will bear in 
mind that his failures are due mostly to his own lack of knowledge and lack of 
skill ; and will truly endeavor to learn and apply the principles laid down in this 
and in the standard works referred to, he will learn after a while to estimate at 
their true value all the pieces of his microscope. (See Ch. X). 



64 LA BORA TOR Y MICROSCOPES [ CH. II 

LABORATORY AND HIGH-SCHOOL COMPOUND 
MICROSCOPES 



\ 121. Optical Parts. — A great deal of beginning work with the microscope in 
biological laboratories is done with simple and inexpensive apparatus. Indeed if 
one contemplates the large classes in the high schools, the universities and med- 
ical schools, it can be readily understood that microscopes costing from $25-50 each 
and magnifying from 25 to 500 diameters, are all that can be expected. But for 
the purpose of modern histological investigation and of advanced microscopical 
work in general, a microscope should have something like the following character : 
Its optical outfit should comprise, (a) dry objectives of 50 mm. (2 in.), 16-18 mm. 
(%in.)and 3 mm. {)/% in.) equivalent focus. There should be present also a 
2 mm. ( T : 2 in. ) or 1.5 mm. (y 1 ^ in.) homogeneous immersion objective. Of oculars 
there should be several of different power. A centering substage condenser, 
and an Abbe camera lucida are also necessities, and a micro-spectroscope and a 
micro-polarizer are very desirable. 

Even in case all the optical parts cannot be obtained in the beginning, it is 
wise to secure a stand upon which all may be used when they are finally secured. 

As to the objectives. The best that can be afforded should be obtained. Cer- 
tainly at the present, the apochromatics stand at the head, although the best 
achromatic objectives approach them very closely. 

\ \ii. Mechanical Parts or Stand. — The stand should be low enongh so that 
it can be used in a vertical position on an ordinary table without inconvenience ; 
it should have a jointed (flexible) pillar for inclination at any angle to the hori- 
zontal. The adjustments for focusing should be two, — a coarse adjustment or 
rapid movement with rack and pinion, and a fine adjustment by means of a mi- 
crometer screw. Both adjustments should move the entire tube of the microscope. 
The body or tube should be short enough for objectives corrected for the short or 
160 millimeter tube-length. It is an advantage to have the draw-tube graduated 
in centimeters and millimeters. The lower end of the draw tube and of the tube 
should each possess a standard screw for objectives (frontispiece). The stage 
should be quite large for the examination of slides with serial sections and other 
large objects. The substage fittings should be so arranged as to enable one to use 
the condenser or to dispense entirely with diaphragms. The condenser mount- 
ing should allow up and down motion. 



STANDARD SIZES RECOMMENDED BY THE ROYAL 
MICROSCOPICAL SOCIETY 



I 123. Society Screw. — Owing to the lack of uniformity in screws for micro- 
scope objectives, the Royal Microscopical Society of London, in 1857, made an 
earnest effort to introduce a standard size. The specifications of this standard are 



CH. II] LABORATORY MICROSCOPES 65 

as follows : "Whitworth thread, i. e., a V shaped thread, sides of thread inclined 
to angle of 55 tp each other, one-sixth of the V depth of the thread being 
rounded off at the top of the thread, and one-sixth of the thread being rounded off 
at the bottom of the thread. Pitch of screw, 36 to the inch ; length of thread on 
object-glass, 0.125 inch ; plain fitting above thread of object-glass, 0.15 inch long, 
to be about the size of the bottom of male thread ; length of thread of nose-piece 
[on the lower end of the tube of the microscope], not less than 0.25 inch ; diam- 
eter of the object-glass screw at the bottom of the screw, 0.7626 inch ; diameter 
of the nose-piece screw at the bottom of the thread, 0.8 inch." 

In order to facilitate the introduction of this universal screw, or as it soon 
came to be called " The Society Screw" the Royal Microscopical Society undertook 
to supply standard taps. From the mechanical difficulty in making these taps 
perfect there soon came to be considerable difference in the "Society Screws," and 
the object of the society in providing a universal screw was partly defeated. 

In 1S84 the American Microscopical Society appointed Mr. Edward Bausch 
and Prof. William A. Rogers upon a committee to correspond with the Royal 
Microscopical Society, with a view to perfecting the standard "Society Screw," 
or of adopting another standard and of perfecting methods by which the screws of 
all makers might be truly uniform. Although this matter was earnestly consid- 
ered at the time by the Royal Microscopical Society, the mechanical difficulties 
were so great that the improvements were abandoned. 

Fortunately, however, during the year (1896) that society again took hold of 
the matter in earnest, and the "Society Screw" is now accurate, and facilities for 
obtaining the standard are so good that there is a reasonable certainty that the 
universal screw for microscopic objectives may be realized. It is astonishing to 
see how widely the "Society Screw" has been adopted. Indeed there is not a 
maker of first-class microscopes in the world who does not supply the objectives 
and stands with the "Society Screw, ' ' and an objective in England or America which 
does not have this screw should be looked upon with suspicion. That is, it is 
either old, cheap, or not the product of one of the great opticians. For the Stand- 
ard, or "Society Screw," see : Trans. Roy. Micr. Soc, 1857, pp. 39-41 ; 1859, PP- 
92-97 ; i860, pp. 103-104. (All to be found in Ouar. Jour. Micr. Sci., o. s., vols. 
VI, VII and VIII). Proc. Amer. Micr. Soc. 1884, p. 274; 1886, p. 199 ; 1893, p. 
38. Journal of the Royal Microscopical Society, August, 1896. 

In this last paper of four pages the matter is very carefully gone over and full 
specifications of the new screw given. It conforms almost exactly with the orig- 
inal standard adopted by the society, but means have been devised by which it 
may be kept standard. 

\ 124. Standard Size Oculars and Substage Condensers. — For a considera- 
tion of these, with measurements, see \ 46, 87. 



MARKERS AND MECHANICAL, STAGES 



Markers are devices to facilitate the finding of some object or part which it is 
especially desired to refer to again or to demonstrate to a class. The mechanical 
stage makes it much easier to follow out a series of objects, to move the slide 
when using high powers, and for complete exploration of a preparation. Most of 
the mechanical stages have scales or scales and verniers by which an object one e 
recorded may be readily found again.. 



66 



LABORATORY MICROSCOPES 



[CH. II 



\ 125. Marker for Preparations. (Figs. 61-66). — This instrument consists of an 
objective-like attachment which may be screwed into the nose ; piece of the micro- 
scope. It bears on its lower end (Figs. 61-3) a small brush and the brush can be 
made more or less eccentric and can be rotated, thus making a larger or smaller 
circle. In using the marker the brush is dipped in colored shellac or other cement 
and when the part of the preparation to be marked is found and put exactly in the 
middle of the field the objective is turned aside and the marker turned into posi- 
tion. The brush is brought carefully in contact with the cover-glass and rotated. 
This will make a delicate ring of the colored cement around the object. Within 
this very small area the desired object can be easily found on any microscope. 
The brush of the marker should be cleaned with 95% alcohol after it is used. 
(Proc. Amer. Micr. Soc, 1894, pp. 112-11S). 




55 



'^W^ 




61. 

Figs. 61-63. Sectional Views of the two Forms of the Marker. 

Fig. 61. The simplest form of marker. It consists of the part SS with the 
milled edge (M). This part bears the society or objective screw for attaching the 
marker to the microscope . R. Rotating part of the marker. This bears the eccen- 
tric brush (B) at its lower end. The brush is on a wire ( W). This wire is eccen- 
tric, and may be made more or less so by bending the wire. The ce?itral dotted 
line coincides with the axis of the microscope. The revolving part is connected 
with the "Society Screw" by the small screw (S ). 

Fig. 62. SS, R, and B. All parts same as with Fig. 61, except that the brush 
is carried by a sliding cylinder the end view being indicated in Fig. 63. 





64 



65 



CH. II] LABORATORY MICROSCOPES 67 




66 

Figs. 64, 65, 66. Specimens Showing the Use of the Marker. 

In Fig. 64 a section of a series ts marked to indicate that this section shows some- 
thing especially well. In Fig. 65 some blood corpuscles showing ingested carbon 
very satisfactorily are surrounded by a minute ring, and in Fig. 66 the lines of a 
micrometer are ringed to facilitate finding the lines. 

\ 126. Pointer in the Ocular. — The Germans have a pointer ocular (Spitzen- 
Okular), an ocular with one or two delicate rods or pointers at the level of the 
real image, that is, at the level of the diaphragm (Figs. 21, 30 D). For the pur- 
poses of demonstrating any particular structure or object in the field, a temporary 
pointer may be easily inserted in any ocular as follows : Remove the eye-lens and 
with a little mucilage or Canada Balsam fasten an eye-lash (cilium) to the diaphragm 
(Fig. 30 D) so that it will project about half way across the opening. If one uses 
this ocular, the pointer will appear in the field and one can place the specimen so 
that the pointer indicates it exactly, as in using a pointer on a diagram or on the 
black-board. It is not known to the author who devised this method. It is cer- 
tainly of the greatest advantage in demonstrating objects like amoebas or white 
blood corpuscles to persons not familiar with them, as the field is liable to have in 
it many other objects which are more easily seen. 

I 127. Mechanical Stage. — For High School and ordinary laboratory work a 
mechanical stage is not needed ; but for much work, especially where high objec- 
tives are used a mechanical stage is of great advantage. It is also advantageous if 
the mechanical stage can be easily removed (see Figs. 67 to 70). The one found 
on the most expensive American and English microscopes for the last twenty years 
and the one now present on the larger continental microscopes, is excellent for 
high powers and preparations of moderate dimensions, but for the study of serial 
sections and large sections or preparations in general, mechanical stages like those 
shown in Figs. 68-69 are more useful. This form of mechanical stage has the 
advantage of giving great lateral and forward and backward motion. It is a mod- 
ification of the mechanical stage of Tolles. The modification consists in doing 
away with the thin plate and having a clamp to catch the ends of the glass slide. 
The slide is then moved on the face of the stage proper. This modification was 
first made by Mayall. It has since been modified by Reichert, Zeiss, Leitz, and 
others in Europe and by the Bausch & Lomb Optical Co., Queen & Co., and the 
Spencer L,ens Co., in America. — Jour. Roy. Micr. Soc, 1885, p. 122. See also 
Zeit. Wiss. Mikroskopie (II), 1885, pp. 289-295 ; 1887 (IV, pp. 25-30). 

Those figured below have the great advantage of ready removal from the stage 
of the microscope, thus leaving it free. They have also the very excellent feature 
that with them one can explore an entire slide full of serial sections, as the sec- 
tions are ordinarily mounted, i. e., under a cover-glass 24X50 mm. 



68 



LABORATORY MICROSCOPES 



{_CH. II 




Fig 67. 




^ 



Fig. 67a. 

Figs. 67, 67a. The removable mechanical stage of the Bausch & Lomb Opti- 
cal Company. In the upper figure it is in position on the stage of the microscope ; 
in the lower figure only the clamping part is in position, the rest having been 
removed to leave the stage of the microscope free . 



CH. //] 



LABOR A TOR Y MICROSCOPES 



69 




Fig. 68. 




Fig. 68a. 



Fig. 68, 68a. Two forms of removable mechanical stage by Leitz. 68 is some- 
what more complex and expensive. Both have the desirable features mentioned 
in \ 12J. 



7o 



LABOR A TORY MICROSCOPES 



[CH. II 







ifflE 



6,0 5 40 30 2 \0 

iiiilmiliiiilmilmiliiiiliiiil i inliiiihmliiiiii, 





Fig. 69. The removable mechanical stage of the Spencer Lens Co. It is a 
modification of a form devised by Winkel. Besides the general features mentioned 
in I i2 7 it has the advantage of fitting any square stage. It is fastened to the stage 
by the clamps shown at the right. Another form is made having one screw on 
the side. 



CH. II] 



LA BORA TOR Y MICROSCOPES 



71 



Fig. 70. Krauss' Method of 
Marking Objectives on a Revolving 
Nose-Piece. 

As seen in the figure, the equiv- 
alent focus of the objective is en- 
graved on the diaphragm above the 
back lens and may be very readily 
seen in rotating the nose-piece. This 
is of great advantage \ as one can see 
what objective is coming into place 
without trouble. It is also an ad- 
vantage in showing where each ob- 
jective belongs when the microscope 
comes from the manufacturers. The 
method is coming into general use. 




FIGURES OF LABORATORY AND HIGH SCHOOL 
MICROSCOPES 



In order that teachers and students may get a good general idea of the appear- 
ance of microscopes of various makers for high school and advanced laboratory 
work a few pictures are appended of the microscopes most used in the United States- 
This has been rendered possible by the courtesy of the manufacturers or import- 
ers. It will be seen that the microscope for the advanced laboratory work is on 
the left and the cheaper one for school and less advanced laboratory work is on 
right. The microscopes are arranged in alphabetical order. 

Laboratory microscopes which will answer nearly all the requirements for 
work in Biology, including Histology, Embryology, Pathology and Bacteriology, 
are listed in the makers catalogs at about $ 100.00. The less expensive micro- 
scopes shown on the right are listed at $25 to $ 45. There is usually a discount of 
10% or more from these prices. Fortunately in the State of New York the State 
pays half for high school apparatus, so that there is no reason why every high 
school should not be properly equipped with microscopes of a good grade. To 
avoid misunderstanding it should be added that the quality of the oculars and 
objectives on the high school microscopes figured is the same as for the best 
laboratory microscopes. The mechanical work also is of excellent quality. 

During the last ten years great vigor has been shown in the microscopical 
world. This has been stimulated largely by the activity in biological science and 
the widespread appreciation of the microscope, not only as a desirable, but as a 
necessary instrument for study and research. The production of the new kinds of 
glass, (Jena glass), and the apochromatic objectives has been a no less potent 
factor in promoting progress. The stadent is advised to write to one or more of 
the opticians for complete catalogs. (See list, p. 2 of cover). 



7 2 



LABORATORY MICROSCOPES 



[CH. II 



Fig. 71 The Bausch & Lomb 
Continental Microscope. BB. 




CH. II] 



LA BORA TORY MICROSCOPES 



73 



Fig. 72. The Baitsch & Lorn 
Continental Microscope, 




74 



LA BORA TORY MICROSCOPES 



\CH. II 




Fig. 73. R. & J. Beck's New Continental Microscope , No. 1152 ( Williams, 
Brown df Earle, Philadelphia). 



CH. //] 



LABOR A TOR Y MICROSCOPES 



75 




Fig. 74. R. & J. Beck's Neiv Continental Microscope, No. 1125 {Williams, 
Brown & Earle, Philadelphia ). 



7 6 



LA BORA TORY MICROSCOPES 



\CH. II 




"wWBiiliiiiiiiiig 
Fig. 75. E. Leitz Microscope il C. ( Wm. Krafjt't, New York). 



CH. II ] 



LABORATORY MICROSCOPES 



11 




Fig. 76. Leitz Microscope HE. It will be noticed that this microscope has no 
joint for inclination ( Wm. Krafft, N. Y.). 



78 



LABORATORY MICROSCOPES 



ten. ii 




Fig. 77. NacheVs Microscope 6 bis. Old model. Nachet is a successor to 
Hartnack who introduced the present "Continental Model" May all, Cantor Lec- 
tures, p. (58 {Franklin Laboratory Supply Co., Boston). 



CH. IT\ 



LABOR A TOR Y MICROSCOPES 



79 




Fig. 78. Nachefs Microscope, No. 11 {Franklin Laboratory Supply Co., Boston). 

This microscope has no joint for inclination, and no rack and pinion for coarse 
adjustment. For coarse adjustment, the tube is pulled up and down with the hands. 
This kind of coarse adjustment was much more common ten years ago than now. 
{See also Fig. 82. ) 



8o 



LA BORA TOR Y MICROSCOPES 



\_CH. II 




Fig. 79. A. Queen & Co' s Continental Microscope, No. II. B. Dust-prooj, 
triple nose-piece . The difference between this and the ordinary form can be seen 
by comparing with Fig. 36. This form of revolving nose-piece has been made for 
many years by Winkel of Goettingen. 



CH. //] 



LABORATORY MICR OS CO PES 




& £15H£L=JI1AN1W& BULfc. 

Fig. 80. Queen & Co's Acme Microscope for Schools 



82 



LABORATORY MICROSCOPES 



\CH. II 



Fig.Si. ReicherVs Nei 
croscope, No. Ill B. (Ric 
& Co., N. Y. and Chicagc 




mm KJm 
ClyL 




CH. II ] 



LABOR A TOR Y MICROSCOPES 



83 




Fig. 82. ReicherVs Microscope, No. V. This has no joint for inclination. 
(Richards & Co., New York and Chicago). 



Fig. 83. The Spencer Lens 
Company's Microscope No. /. 
This has the standard size , Con- 
tinental oculars, and the object- 
ives and oculars have the equiv- 
alent focus in millimeters. 




Fig. 84. The Spencer Lens 
Company's Microscope, No. 2. 




LABORATORY MICROSCOPES 



[CH. // 




Fig. 85. Watson & Sons, Edinburgh Students' Microscope {Stand G). This 
is a good representative of the tripod-base, English models of to-day. It is in gen- 
eral like the Powell and Lealand stands which have held their position with the 
foremost English microscopists for the last 4.0 years. [See Carpenter-Dallinger, 
p. 172. ) 

Microscopes with tripod base something after this pattern are now being 
made on the Continent. 

Attention is called to the sub-stage for the condenser . It possesses centering 
screws so that any apparatus used in it may be accurately centered. It is to be 
hoped that all microscopes of this grade will soon be supplied with a centering 
sub-stage. 



CH. II] 



LA BO J? A TOR Y MICROSCOPES 



87 




Fig. 86. Zeiss Micro scop e~i & with Mechanical Stage. This figure from Zeiss' 
Catalog No. jo, represents the Continental Model of Microscope in its most perfect 
form. 

K. Milled head of the screw for the lateral movements of the stage. 

L. Screw for fixing the laterally moving mechanism of the stage. By un- 
screwing this the laterally moving part may be removed, leaving the plain stage. 
W. Screw for moving the stage forward and backward. 



LABOR A TOR Y MICROSCOPES 



\CH. II 




Fig. 87, Zentmeyer's Microscope, No. V. 



CH. /I] 



LA BORA TORY MICROSCOPES 



89 




Fig. 88. Zentmayer 's Microscope , No. IV. 



CHAPTER III 



INTERPRETATION OF APPEARANCES 



APPARATUS AND MATERIAL FOR CHAPTER III 

I 
A laboratory, compound microscope ('$ 121 ); Preparation of fly's wing ; 50 per 
cent, glycerin; Slides and covers; Preparation of letters in stairs (Fig. 89); 
Mucilage for air-bubbles and olive or clove oil for oil-globules (§ 136-139). Solid 
glass rod, and glass tube (§ 144-146); Collodion (g 146); Carmine, India ink, or 
lamp black (| 148-150); Frog, castor oil and micro-polariscope a ( § 152). 

INTERPRETATION OF APPEARANCES UNDER THE MICROSCOPE 

§ 129. General Remarks. — The experiments in this chapter are 
given secondarily for drill in manipulation, but primarily so that the 
student may not be led into error or be puzzled by appearances which 
are constantly met with in microscopical investigation. Anyone can 
look into a microscope, but it is quite another matter to interpret cor- 
rectly the meaning of the appearances seen. 

It is especially important to remember that the more of the relations 
of any object are known, the truer is the comprehension of the object. 
In microscopical investigation ever)' object should be scrutinized from 
all sides and under all conditions in which it is likely to occur in nature 
and in microscopical investigation. It is best also to begin with objects 
of considerable size whose character is well known, to look at them 
carefully with the unaided eye so as to see them as wholes and in their 
natural setting ; then a low power is used, and so on, step by step until 
the highest power available has been employed. One will in this way 
see less and less of the object as a whole, but every increase in magnifi- 
cation will give increased prominence to detail, detail which might be 
meaningless when taken alone and independent of the object as a 
whole. The pertinence of this advice will be appreciated when the 
student undertakes to solve the problems of histology ; for even after all 
the years of incessant labor spent in trying to make out the structure 
of man and the lower animals, many details are still in doubt, the 
same visual appearances being quite differently interpreted by eminent 
observers. 



CH. Ill] INTERPRETATION OF APPEARANCES 91 

Appearances which seem perfectly unmistakable with a low power 
may be found erroneous or very inadequate, for details of structure that 
were indistinguishable with the low power may become perfectly evi- 
dent with a higher power or a more perfect objective. Indeed the prob- 
lems of microscopic structure appear to become ever more complex, for 
difficulties overcome by improvements in the microscope simply give 
place to new difficulties, which in some cases render the subject more 
obscure than it appeared to be with the less perfect appliances. 

The need of the most careful observation and constant watchful- 
ness lest the appearances may be deceptive are thus admirably stated 
by Dallinger (see Carpenter-Dallinger, p. 427): "The correctness of 
the conclusions which the microscopist will draw 7 regarding the nature 
of any object from the visual appearances which it presents to him 
when examined in the various modes now specified will necessarily 
depend in a great degree upon his previous experience in microscopic 
observation and upon his knowledge of the class of bodies to which 
the particular specimen may belong. Not only are observations of 
any kind liable to certain fallacies arising out of the previous notions 
which the observer may entertain in regard to the constitution of the 
objects' or the nature of the actions to which his attention is directed, 
but even the most practiced observer is apt to take no note of such 
phenomena as his mind is not prepared to appreciate. Errors and im- 
perfections of this kind can only be corrected, it is obvious, by general 
advance in scientific knowledge ; but the history of them affords a use- 
ful warning against hasty conclusions drawn from a too cursor)' exam- 
ination. If the history of almost any scientific investigation were 
full}' made known it would generally appear that the stability and 
completeness of the conclusions finally arrived at had been only 
attained after many modifications, or even entire alterations, of doctrine. 
And it is therefore of such great importance as to be almost essential 
to the correctness of our conclusions that they should not be finally 
formed and announced until they have been tested in every conceivable 
mode. It is due to science that it should be burdened with as few false 
facts [artifacts] and false doctrines as possible. It is due to other 
truth-seekers that they should not be misled, to the great waste of 
their time and pains, by our errors. And it is due to ourselves that 
we should not commit our reputation to the chance of impairment by 
the premature formation and publication of conclusions which may be 
at once reversed by other observers better informed than ourselves, or 
may be proved fallacious at some future time, perhaps even by our 



92 INTERPRETATION OF APPEARANCES \_CH. Ill 

own more extended and careful researches. The suspension of the judg- 
ment whenever there seems room for doubt is a lesson inculcated hy all 
those philosophers who have gained the highest repute for practical 
wisdom ; and it is one which the mieroscopist cannot too soon learn or 
too constantly practice." 

For these experiments no condenser is to be used except where 
specifically indicated. 

§ 130. Dust or Cloudiness on the Ocular. — Employ the 16 
mm. ( 2 3 in.) objective, low ocular, and fly's wing as object. 

Unscrew the field-lens and put some particles of lint from dark 
cloth on its upper surface. Replace the field-lens and put the ocular 
in position (§ 48). Eight the field well and focus sharply. The im- 
age will be clear, but part of the field will be obscured by the irregular 
outline of the particles of lint. Move the object to make sure this 
appearance is not due to it. 

Grasp the ocular b3 T the milled ring, just above the tube of the 
microscope, and rotate it. The irregular objects will rotate with the 
ocular. Cloudiness or particles of dust on any part of the ocular may 
be detected in this way. 

§ 131. Dust or Cloudiness on the Objective. — Employ the 
same ocular and objective as before and the fly's wing as object. Focus 
and light well, and observe carefully the appearance. Rub gfycerin 
on one side of a slide near the end. Hold the clean side of this end 
close against the objective. The image will be obscured, and cannot 
be made clear by focusing. Then use a clean slide and the image may 
be made clear by elevating the tube slightly. The obscurity produced 
in this way is like that caused hy clouding the front-lens of the objec- 
tive. Dust would make a dark patch on the image that would remain 
stationary while the object or ocular is moved. 

If a small diaphragm is employed and it is close to the object, 
only the central part of the field will be illuminated, and around the 
small light circle will be seen a dark ring (Fig. 42). If the diaphragm 
is lowered or a sufficiently large one employed the entire field will be 
lighted. 

§ 132. Relative Position of Objects or parts of the same 
object. The general rule is that objects highest up come into focus 
last in focusing vc$,fi7'st in focusing down. 

§ 133. Objects having Plane or Irregular Outlines. — As object 
use three printed letters in stairs mounted in Canada balsam (Fig. 89). 
The first letter is placed directly upon the slide, and covered with a 





b 




c 




d 


.s' / 



CH. Ill] INTERPRETATION OF APPEARANCES 93 

small piece of glass about as thick as a slide. The second letter is 
placed upon this and covered in like manner. The third letter is placed 
upon the second thick cover and covered with an ordinary cover-glass. 
The letters should be as near together as possible, but not over-lapping. 
Employ the same ocular and objective as above (§ 130). 

Fig. 89. letters mounted in stairs to 
show the order of coming into focus. 

a, b, c, d. The various letters indi- 
cated by the oblique row of black marks in 
sectional view. Slide. The glass slide on which the letters are mounted. 

Lower the tube till the objective almost touches the top letter, then 
look into the microscope, and slowly focus up. The lowest letter will 
first appear, and then, as it disappears, the middle one will appear, and 
so on. Focus down, and the top letter will first appear, then the mid- 
dle one, etc. The relative position of objects is determined exactly in 
this way in practical work. 

For example, if one has a micrometer ruled on a cover-glass 15-25 
hundredths mm. thick, it is not easy to determine with the naked eye 
which is the ruled surface. But if one puts the micrometer under a 
microscope and uses a 3 mm.(^ in.) objective, it is easily determined. 
The cover should be laid on a slide and focused till the lines are sharp. 
Now, without changing the focus in the least turn the cover over. If 
it is necessary to focus up to get the lines of the micrometer sharp, the 
lines are on the upper side. If one must focus down, the lines are on 
the under surface. With a thin cover and delicate lines this method of 
determining the position of the rulings is of considerable practical 
importance. 

§ 134. Determination of the Form of Objects. — The procedure 
is exactly as for the determination of the fo^m of large objects. That 
is, one must examine the various aspects. For example, if one were 
placed in front of a wall of some kind he could not tell whether it was 
a simple wall or whether it was one side of a building unless in some 
way he could see more than the face of the wall. In other words, in 
order to get a correct notion of any body, one must examine more than 
one dimension, — two for plane surfaces, three for solids. So for micro- 
scopic objects, one must in some way examine more than one face. To 
do this with small bodies in a liquid the bodies may be made to roll 
over by pressing on one edge of the cover-glass. And in rolling over 
the various aspects are presented to the observer. With solid bodies, 



94 INTERPRETATION OF APPEARANCES \CH. Ill 

like the various organs, correct notions of the form of the elements can 
be determined by studying sections cut at right angles to each other. 
The methods of getting the elements to roll over, and of sectioning in 
different planes are in constant use in Histology, and the microscopist 
who neglects to see all sides of the tissue elements has a very inade- 
quate and often a very erroneous conception of their true form. 

§ 135. Transparent Objects having Curved Outlines. — The 
success of these experiments will depend entirely upon the care and skill 
used in preparing the objects, in lighting, and in focusing. 

Employ a 3 mm. (}£ in.) or higher objective and a high ocular for 
all the experiments. It may be necessary to shade the object (§ 109) 
to get satisfactory results. When a diaphragm is used the opening 
should be small and it should be close to the object. 

§ 136. Air Bubbles. — Prepare these by placing a drop of thin 
mucilage on the center of a slide and beating it with a scalpel blade 
until the mucilage looks milky from the inclusion of air bubbles. Put 
on a cover-glass but do not press it down. 

Fig. 90. Diagram show- 
TVj^j^^c ^ pp ing how to place a cover- 

glass upon an object with the 
forceps. 




^m, 



§ 137. Air Bubbles with Central Illumination. — Shade the 
object ; and with the plane mirror, light the field with central light 
(Fig. 23). 

Search the preparation until an air bubble is found appearing 
about 1 mm. in diameter, get it into the center of the field, and if the 
light is central the air bubble will appear with a wide, dark, circular 
margin and a small brig*ht center. If the bright spot is not in the 
center, adjust the mirror until it is. 

This is one of the simplest and surest methods of telling when the 
light is central or axial when no condenser is used (§ 65). 

Focus both up and down, noting that, in focusing up, the central 
spot becomes very clear and the black ring very sharp. On elevating 
the tube of the microscope still more the center becomes dim, and the 
whole bubble loses its sharpness of outline. 

§ 13S. Air Bubbles with Oblique Illumination. — Remove the 
sub-stage of the microscope and all the diaphragms. Swing the mirror 
so that the ra} T s may be sent very obliquely upon the object (Fig. 23, 



CH. Ill} INTERPRETATION OF APPEARANCES 95 

C). The bright spot will appear no longer in the center but on the 
side away ft vm the mirror (Fig. yi). 

§ 139. Oil Globules. — Prepare these by beating a small drop of 
clove oil with mucilage on a slide and covering as directed for air bub- 
bles (§ 137), or use a drop of milk. 

§ 140. Oil Globules with Central Illumination. — Use the same 
diaphragm and light as above (§ 137). Find an oil globule appearing 
about 1 mm. in diameter. If the light is central a bright spot will ap- 
pear in the center as with air. Focus up and down as with air, and 
note that the bright center of the oil globule is clearest last in focus- 
ing up. 

Fig. 91. Very small Globule of Oil (O) and an Air Bubble 
(A) seen by Oblique Light. The arrow indicates the direction of 
the light rays. 

§ 141. Oil Globules with Oblique Illumination.— 
Remove the sub-stage, etc., as above, and swing the mir- 
ror to one side and light with oblique light. The bright 
spot will be eccentric, and will appear to be on the same 
side as the mirror (Fig. 91). 

§ 142. Oil and Air Together. — Make a prepara- 
tion exactly as described for air bubbles (§ 136), and add 
at one edge a little of the mixture of oil and mucilage 
(§ J 39) ! cover and examine. 

The sub-stage need not be used in this experiment. Search the 
preparation until an air bubble and an oil globule, each appearing 
about 1 mm. in diameter, are found in the same field of view. Light 
first with central light, and note that, in focusing up, the air bubble 
comes into focus first and that the central spot is smaller than that of 
the oil globule. Then, of course, the black ring will be wider in the 
air bubble than in the oil globule. Make the light oblique. The 
bright spot in the air bubble will move away from the mirror while 
that in the oil globule will move toward it. See Fig. 91.* 

§ 143. Air and Oil by Reflected Light. — Cover the diaphragm 
or mirror so that no transmitted light (§ 64) can reach the preparation, 
using the same preparation as in § 142. The oil and air will appear 
like globules of silver on a dark ground. The part that was darkest in 




*It should be remembered that the image in the compound microscope is 
inverted (Fig. 21), hence the bright spot realty moves toward the mirror for air, 
and away from it for oil. 



96 INTERPRETATION OF APPEARANCES [CH. Ill 

each will be lightest, and the bright central spot will be somewhat 
dark.* 

§ 144. Distinctness of Outline. — In refraction images this 
depends on the difference between the refractive power of a body and 
that of the medium which surrounds it. The oil and air were very 
distinct in outline as both differ greatly in refractive power from the 
medium which surrounds them, the oil being more refractive than the 
mucilage and the air less. (Figs. 54-56.) 

Place a fragment of a cover-glass on a clean slide, and cover it 
(see under mounting). The outline will be distinct with the unaided 
eye. Use it as object and employ the 16 mm. (Jfi in.) objective and 
high ocular. Light with central light. The fragment will be outlined 
by a dark band. Put a drop of water at the edge of the cover-glass. 
It will run in and immerse the fragment. The outline will remain dis- 
tinct, but the dark band will be somewhat narrower. Remove the 
cover-glass, wipe it dry, and wipe the fragment and slide dry also. 
Put a drop of 50% glycerin on the middle of the slide and mount the 
fragment of cover-glass in that. The dark contour will be much nar- 
rower than before. 

Draw a solid glass rod out to a fine thread. Mount one piece in 
air, and the other in 50% glycerin. Put a cover-glass on each. Em- 
ploy the same optical arrangement as before. Examine the one in air 
first. There will be seen a narrow, bright band, with a wide, dark 
band on each side. 

The one in glycerin will show a much wider bright central band, 
with the dark borders correspondingly narrow (Fig. 92, b). The dark 
contour depends also on the numerical aperture of the objective — being 
wider with low apertures. This can be readily understood when it is 
remembered that the greater the aperture the more oblique the rays of 
light that can be received, and the dark band simply represents an 
area in which the rays are so greatly bent or refracted (Figs. 54-56) 
that the}' cannot enter the objective and contribute to the formation of 
the image ; the edges are dark simply because no light from them 
reaches the observer. 



*It is possible to distinguish oil and air optically, as described above, only 
when quite high powers are used and very small bubbles are selected for observa- 
tion. If a 16 mm. {% in.) is used instead of a 3 mm. {y& in.) objective, the ap- 
pearances will vary considerably from that given above for the higher power. It 
is well to use a low as well as a high power. Marked differences will also be 
seen in the appearances with objectives of small and of large aperture. 




CH.III] INTERPRETATION OF APPEARANCES 97 

Fig. 92. Solid glass rod showing the 
appearance when viewed with transmit- 
ted, central light, and with an objective 
of medium aperture. 

a. Mounted in air. b. Mounted in 50 per cent, glycerin. 

If the glass rod or any other object were mounted in a medium of 
the same color and refractive power, it could not be distinguished from 
the medium.* 

A very striking and satisfactory demonstration may be made by 
painting a zone or band of eosin or other transparent color on a solid 
glass rod, and immersing the rod in a test tube or vial of cedar oil, 
clove oil or turpentine. Above the liquid the glass rod is very evident, 
as it is also at the colored zone, but at other levels it can hardly be 
seen in the liquid. 

§ 145. Highly Refractive. — This expression is often used in de- 
scribing microscopic objects, (medullated nerve fibers, for example), 
and means that the object will appear to be bordered by a wide, dark 
margin when it is viewed by transmitted light. And from the above 
(§ 144), it would be known that the refractive power of the object, and 
the medium in which it was mounted must differ considerably. 

§ 146. Doubly Contoured. — This means that the object is 
bounded by two, usually parallel dark lines with a lighter band between 
them. In other words, the object is bordered by (1) a dark line, (2) a 
light band, and (3) a second dark line (Fig. 93). 

This ma}' be demonstrated by coating a fine glass rod (§ 144) with 
one or more coats of collodion or celloidin and allowing it to dry, and 
then mounting in 50% glycerin as above. Employ a 3 mm.(^ in.) or 
higher objective, light with transmitted light, and it will be seen that 
where the glycerin touches the collodion coating there is a dark line — 
next this is a light band, and finally there is a second dark line where 
the collodion is in contact with the 
glass rod.* (Fig. 93). 

Fig. 93. Solid glass rod coated with col-\ 
lodion to show a double contour. Toward ' 
one end the collodion had gathered in a fusi- 
form drop. 




*Some of the rods have air bubbles in them, and then there results a capillary 
tube when they are drawn out. It is well to draw out a glass tube into a fine 
thread and examine it as described. The central cavity makes the experiment 
much more complex. 



98 INTERPRETATION OF APPEARANCES [CH. Ill 

§ 147. Optical Section. — This is the appearance obtained in 
examining transparent or nearly transparent objects with a microscope 
when some plane below the upper surface of the object is in focus. 
The upper part of the object which is out of focus obscures the image 
but slightl}*. By changing the position of the objective or object, a 
different plane will be in focus and a different optical section obtained. 
The most satisfactory optical sections are obtained with high objectives 
having large aperture. 

Nearly all the transparent objects studied may be viewed in optical 
section. A striking example will be found in studying mammalian 
red blood-corpuscles on edge. The experiments with the solid glass 
rods (Fig. 92) furnish excellent and striking examples of optical 
sections. 

§ 148. Currents in Liquids. — Employ the 16 mm. (^ in.) ob- 
jective, and as object put a few particles of carmine on the middle of a 
slide, and add a drop of water. Grind the carmine well with a scalpel 
blade, and then cover it. If the microscope is inclined, a current will 
be produced in the water, and the particles of carmine will be carried 
along by it. Note that the particles seem to flow up instead of down — 
why is this ? 

Lamp-black rubbed in water containing a little mucilage answers 
well for this experiment. 

§ 149. Velocity Under the Microscope. — In studying currents 
or the movement of living things under the microscope, one should 
not forget that the apparent velocity is as unlike the real velocity as 
the apparent size is unlike the real size. If one consults Fig. 37 it 
will be seen that the actual size of the field of the microscope with the 
different objectives and oculars is inversely as the magnification. That 
is, with great magnification only a small area can be seen. The field 
appears to be large, however, and if any object moves across the field 
it ma}* appear to move with great rapidity, whereas if one measures 
the actual distance passed and notes the time, it will be seen that the 
actual motion is quite slow. One should keep this in mind in stud)'- 
ing the circulation of the blood. The truth of what has just been 
said can be easily demonstrated in studying the circulation in the gills 



*The collodion used is a 6% solution of gun cotton in equal parts of sulphuric 
ether and 95% alcohol. It is well to dip the rod two or three times in the collo- 
dion and to hold it vertically while drying. The collodion will gather in drops, 
and one will see the difference between a thick and a thin membranous covering. 
(Fig- 93)- 



CH. Ill ] INTER PRE TA TION OF A PRE A RANCES 99 

of Necturus, or in the frog's foot, by using first a low power in which 
the field is actually of considerable diameter (Fig. 37, Table, § 51) and 
then using a high power. With the high power the apparent motion 
will appear much more rapid. For spiral, serpentine and other forms 
of motion, see Carpenter-Dallinger, p. 433. 

§ 150. Pedesis or Brownian Movement. — Employ the same 
object as above, but a 3 mm. (}i in.) or higher objective in place of 
the 16 mm. Make the body of the microscope vertical, so that there 
ma}* be no currents produced. Use a small diaphragm and light the 
field well. Focus and there will be seen in the field large motionless 
masses, and between them small masses in constant motion. This is 
an indefinite, dancing or oscillating motion. 

This indefinite but continuous motion of small particles in a liquid 
is called Pe-de sis or Brownian movement. Also, but improperly, molec- 
iilar movement, from the smallness of the particles. 

The motion is increased by adding a-litfe gum arabic solution or 
a slight amount of silicate of soda or soap ; sulphuric acid and various 
saline compounds retard or check the motion. One of the best objects 
is lamp-black ground up with a little gum arabic. Carmine prepared 
in the same way, or simply in water, is excellent ; and very finely 
powdered pumice-stone in water has for many years been a favorite 
object. 

Pedesis is exhibited by all solid matter if it is finely enough di- 
vided and in a suitable liquid. In the minds of most, no adequate 
explanation has yet been offered. See Carpenter-Dallinger, p. 431 ; 
Beale, p. 195 ; Jevons in Quart. Jour. Science, n. s., Vol. VIII (1878), 
p. 167. In 1894, Meade Bache published a paper in the Proc. Amer. 
Philos. Soc, Vol. XXXIII, pp. 163-167, entitled "The Secret of 
the Brownian Movement." This paper is suggestive if not very 
satisfactory. 

For the orginal account of this see Robert Brown, "Botanical 
appendix to Captain King's voyage to Australia," Vol. II, p. 534. 
(1826). 

See also Dr. C. Aug. Sigm. Schultze, "Mikroskopische Unter- 
suchungen fiber des Herren Robert Brown Entdeckung lebender, selbst 
im Feuer unzerstorbarer Theilchen in alien Korpern." From "Die 
Gesellschaft fur Betorderung der Naturwissenschaften zu Freiburg. ' ' 
1828. 

Compare the pedetic motion with that of a current hy slightly in- 
clining the tube of the microscope. The small particles will continue 



I oo IN TERPRE TA TION OF A PRE A RA NCES [ CH. Ill 

their independent leaping movements while they are carried along by 
the current. The pedetic motion makes it difficult to obtain good 
photographs of milk globules and other small particles. The difficulty 
may be overcome by mixing the milk with a very weak solution of 
gelatin and allowing it to cool (see Ch. IX). 

§ 151. Demonstration of Pedesis with the Polarizing Micro- 
scope (Ch. VI). — The following demonstration shows conclusively 
that the pedetic motion is real and not illusive. (Ranvier, p. 173.) 

Open the abdomen of a dead frog (an alcoholic or formalin 
specimen is satisfactory). Turn the viscera to one side and observe 
the small, whitish masses at the emergence of the spinal nerves. With 
fine forceps remove one of these and place it on the middle of a clean 
slide. Add a drop of water, or of water containing a little gum arabic. 
Rub the white mass around in the drop of liquid and soon the liquid 
will have a milky appearance. Remove the white mass, place a cover- 
glass on the milky liquid and seal the cover by painting a ring of 
castor oil all around it, half the ring being on the slide and half 
on the cover-glass. This is to avoid the production of currents 
evaporation. 

Put the preparation under the microscope and examine with, first ; 
a low then a high power (3 mm. or /s in.). In, the field will be seen 
multitudes of crystals of carbonate of lime ; the larger crystals are 
motionless but the smallest ones exhibit marked pedetic movement. 

Use the micro-polariscope, light with great care and exclude all 
adventitious light from the microscope by shading the object (§ 109) and 
also by shading the eye. Focus sharply and observe the pedetic motion 
of the small particles, then cross the polarizer and analyzer, that is, 
turn one or the other until the field is dark. Part of the large motion- 
less crystals will shine continuously and a part will remain dark, but 
small crystals between the large ones will shine for an instant, then 
disappear, only to appear again the next instant. This demonstration 
is believed to furnish absolute proof that the pedetic movement is real 
and not illusory. 

§ 152. Muscae Volitantes. — These specks or filaments in the 
eyes due to minute shreds or opacities of the vitreous sometimes appear 
as part of the object as they are projected into the field of vision. They 
may be seen by looking into the well lighted microscope when there is 
no object under the microscope. They may also be seen by looking 
at brightly illuminated snow or other white surface. By studying 
them carefully it will be seen that they are somewhat movable and float 




CH. Ill ] INTER PRE TA TION OF A PPEARANCES I o I 

across the field of vision, and thus do not remain in one position as do 
the objects under observation. Furthermore, one may, by taking a 
little pains, familiarize himself with the special forms in his own eyes 
so that the more conspicuous at least may be instantly recognized. 

§153. In addition to the above experiments it is very strongly 
recommended that the student follow the advice of Beale, p. 248, and 
examine first with a low then with a higher power, mounted dry, then 
in water, lighted with reflected light, then with transmitted light, the 
following : Potato, wheat, rice, and corn starch, easily obtained by 
scraping the potato and the grains mentioned ; bread crumbs ; portions 
of feather. Portions of feather accidentally present in histological 
preparations have been mistaken for lymphatic vessels (Beale, 288). 
Fibers of cotton, linen and silk. Textile fibers accidentally present 
have been considered nerve fibers, etc. Human and animal hairs. 
Study with especial care hairs from various parts of the body of the 
animals used for dissection in the laboratory where you work. These 
are liable to be present in histological preparations, and unless their 
character is understood there is chance for much confusion and erro- 
neous interpretation. The scales of butterflies and moths, especially 
the common clothes moth. The dust swept from carpeted and wood 
floors. Tea leaves and coffee grounds. Dust found in living rooms 
and places not frequently dusted. In the last will be found a regular 
museum of object's. 

For figures (photo- micrographs, etc.) of the various forms of starch, 
see Bulletin No. 13 of the Chemical Division of the U. S. Department 
af Agriculture. For Hair and Wool, see Bulletin of the National Asso- 
ciation of Wool Growers, 1875, P- 47°> Proc Amer. Micr. Soc, 1884, 
pp. 65-68. Herzfeld, translated by Salter. — The technical testing of 
yarns and textile fabrics, London, 1898. 

For different appearances due to the illuminator, see Nelson, in 
Jour. Roy, Micr. Soc, 1891, pp. 90-105 ; and for the illusory appear- 
ances due to diffraction phenomena, see Carpenter-Dallinger, p. 434. 
Mercer. Trans. Amer. Micr. Soc, pp. 321-396. 

If it is necessary to see all sides of an ordinary gross object, and 
to observe it with varying illumination and under various conditions of 
temperature, moisture, etc., in order to obtain a fairly accurate and 
satisfactory knowledge of it, so much the more is it necessary not to be 
satisfied in microscopical observation until every means of investigation 
and verification has been called into service, and then of the image 
that falls upon the retina, only such details will be noted as the brain 
behind the eye is ready to appreciate. 



102 



INTERPRETATION OF APPEARANCES 



[CH. Ill 



To summarize this chapter and leave with the beginning student 
the result of the experience of many eminent workers : 

i. Get all the information possible with the unaided eye. See 
the whole object and all sides of it, so far as possible. 

2. Examine the preparation with a simple microscope in the same 
thorough way for additional detail. 

3. Use a low power of the compound microscope. 

4. Use a higher power. 

5. Use the highest power available and applicable. In this way 
one sees the object as a whole and progressively more and more details. 
Then as the object is viewed from two or more aspects, something like 
a correct notion ma} 7 be gained of its form and structure. 




THE MICROSCOPE IN SECTION 



1. Positive ocular. 

2. Draw-tube. 

3. Maiu tube or body. 

4-5. Society screws in the 
Jdraw-tube and body. 

6. Objective in position. 

7. Stage. 

S. Spring for holding 

slides. 
9. Sub-stage condenser. 
- 10. Iris diaphragm. 



n. Plane and concave mir- 
ror. 

12. Horse-shoe base. 

13. Rack and pinion for 

condenser. 

14. Flexible pillar. 

15. Spiral spring of fine ad- 

justment. 

16. Fine adjustment 

17. Coarse adjustment. 



CHAPTER IV 



MAGNIFICATION AND MICROMETRY 



APPARATUS AND MATERIAL FOR THIS CHAPTER 

Simple and compound microscope (I 156, 158); Steel scale or rule divided to 
millimeters and Aths ; Block for magnifier and compound microscope ($ 156, 160); 
Dividers (§ 156, 160); Stage micrometer ($ 159); Wollaston camera lucida ( \ 160); 
Ocular screw-micrometers (Figs. 106-107); Micrometer ocular (Figs. 104-105). 
Abbe camera lucida (Fig. 101). Necturus red blood corpuscles (I 168). 

§ 154. The Magnification, Amplification or Magnifying Power 
of a simple or compound microscope is the ratio between the real and 
the apparent size of the object examined. The apparent size is ob- 
tained by measuring the virtual image (Figs. 21, 38). The object for 
determining magnification must be of known length and is designated 
a micrometer (§ 159). In practice a virtual image is measured by the 
aid of some form of camera lucida (Figs. 97, 101), or by double vision 
(§ 156). As the length of the object is known, the magnification is 
easily determined by dividing the apparent size of the image by the 
actual size of the object. For example, if the virtual image measures 
40 mm. and the object magnified, 2 mm., the amplification must be 
40 -4- 2 = 20, that is, the apparent size is 20 fold greater than the real 
size. 

Magnification is expressed in diameters or times linear, that is, but 
one dimension is considered. In giving the scale at which a micro- 
scopical or histological drawing is made, the word magnification is fre- 
quently indicated by the sign of multiplication thus : X 450, upon a 
drawing would mean that the figure or drawing is 450 times as large 
as the object. 

§ 155. Magnification of Real Images. — In this case the mag- 
nification is the ratio between the size of the real image and the size of 
the object, and the size of the, real image can be measured directly. By 
recalling the work on the function of an objective (§ 53), it will be 
remembered that it forms a real image on the ground glass placed on 
the top of the tube, and that this real image could be looked at with the 



104 



MAGNIFICATION AND MICROMETRY 



[CH. IV 



eye or measured as if it were an actual object. For example, suppose 
the object were three millimeters long and its image on the ground glass 
measured 15 mm., then the magnification must be, 15 -5- 3 = 5, that is, 
the real image is 5 times as long as the object. The real images seen 
in photography are mostly smaller than the objects, but the magnifica- 
tion is designated in the same way by dividing the size of the real im- 
age measured on the ground glass by the size of the object. For 
example, if the object is 400 millimeters long and its image on the 
ground glass is 25 mm. long, the ratio is 25-^40Q=- 1 1 g. That is, the 
image is y^th as long as the object and is not magnified but reduced. 
In marking negatives, as with drawings, the sign of multiplication is 
put before the ratio, and in the example the designation would be 

XyVth. 

MAGNIFICATION OF A SIMPLE MICROSCOPE 



§ 156. The Magnification of 
a Simple Microscope is the ratio 
between the object magnified (Fig. 
16, A'B'), and the virtual image 
(A 3 B 3 ) . To obtain the size of this 
virtual image place the tripod mag- 
nifier near the edge of a support of 
such a height that the distance 
from the upper surface of the mag- 
nifier to the table is 250 millimeters. 



Fig. 94. Tripod Magnifier. 

As object, place a scale of some kind ruled in millimeters on the 
support under the magnifier. Put some white paper on the table at 
the base of the support and on the side facing the light. 





Fig. 95. Ten Centimeter Rule. The upper edge is divided into millimeters, 
the lower into centimeters at the left and half centimeters at the right. 

Close one eye, and hold the head so that the other will be near the 
upper surface of the lens. Focus if necessary to make the image clear 



CH. IV] MA GNIFICA TION AND MICRO ME TRY 105 

(§ 11). Open the closed eye and the image of the rule will appear as 
if on the paper at the base of the support. Hold the head very still, 
and with dividers get the distance between any two lines of the image. 
This is the so-called method of double vision in which the microscope 
image is seen with one eye and the dividers with the other, the two 
images appearing to be fused in a single visual field. 

§ 157. Measuring the Spread of Dividers. — This should be 
done on a steel scale divided to millimeters and iths. 

As \ mm. cannot be seen plainly by the unaided eye, place one 
arm of the dividers at a centimeter line, and with the tripod magnifier 
count the number of spaces on the rule included between the points of 
the dividers. The magnifier simply makes it easy to count the spaces 
on the rule included between the points of the dividers — it does not, of 
course, increase the number of spaces or change their value. 

As the distance between any two lines of the image of the scale 
gives the size of the virtual image (Fig. 16, A :5 B 3 ), and as the size of 
the object is known, the magnification is determined by dividing the 
size of the image by the size of the object. Thus, suppose the distance 
between the two lines of the image is measured by the dividers and 
found on the steel scale to be 15 millimeters, and the actual size of the 
space between the two lines of the object is 2 millimeters, then the 
magnification must be 15^2=7^. That is, the image is 7^2 times as 
long or wide as the object. In this case the image is said to be 
magnified 7^ diameters, or 7^ times linear. 

The magnification of any simple magnifier may be determined 
experimentally in the way described for the tripod. 

MAGNIFICATION OF A COMPOUND MICROSCOPE 

§ 158. The Magnification of a Compound Microscope is the 
ratio between the final or virtual image (Fig. 21, B 3 A 3 ), and the object 
magnified (A B). 

The determination of the magnification of a compound microscope 
may be made as with a simple microscope (§ 156), but this is very 
fatiguing and unsatisfactory. 

£ 159. Stage, Object or Objective Micrometer. — For deter- 
mining the magnification of a compound microscope and for the 
purpose of micrometry, it is necessary to have a finely divided scale 
or rule on glass or on metal. Such a finely divided scale is called a 
micrometer, and for ordinary work one mounted on a glass slide 
(1X3 in, 25 X 76 mm.) is most convenient. 



1 06 MA GN1FICA TION AND MICROME TRY \CH. IV 

The spaces between the lines should be y 1 -^ and T ^ nam. (or if in 
inches, y^ and toVo m -) Micrometers are sometimes ruled on the 
slide, but more satisfactorily on a cover-glass of known thickness, 
preferably 0.15-0. 18 mm. The covers should be perfectly clean before 
the ruling, and afterwards simply dusted off with a camel's hair 
duster, and then mounted, lines downward over a shellac or other 
good cell. (See Ch. VII). If one rubs the lines the edges of the 
furrow made by the diamond are liable to be rounded and the sharp- 
ness of the micrometer is lost. If the lines are on the slide and un- 
covered one cannot use the micrometer with an oil immersion, as the 
oil obliterates the lines. Cleaning the slide makes the lines less sharp 
as stated. If the lines are very coarse, it is an advantage to fill them 
with plumbago. This may be done either with some very fine plum- 
bago on the end of a soft cork, or by using an exceedingly soft lead 
pencil. Lines properly filled may be covered with balsam and a cover- 
glass as in ordinary balsam mounting (Ch. VII). 

§ 160. Determination of Magnification. — This is most readily 
accomplished b}^ the use of some form of camera lucida (Ch. V), that 
of Wollaston being most convenient as it may be used for all powers, 
and the determination of the standard distance of 250 millimeters at 
which to measure the images is very readily determined (Fig. 97, § 162)- 

Employ the 16 mm. { 2 /i in.) objective and a 37 mm. (or X 8 ocu- 
lar with a stage micrometer as object. For this power the -^ mm. 
spaces of the micrometer should be used as object. Focus sharply. 

Fig. 96. Diagram of a stage micrometer, 
with a ring on the lines to facilitate finding them. 

It is somewhat difficult to find the mi- 
crometer lines. To avoid this it is well to 
have a small ring enclosing some of the 
micrometer lines (Fig. 96). The light must also be carefully regu- 
lated. If too much light is used, i. e., too large an aperture, the lines 
will be drowned in the light. In focusing with the high powers be 
very careful. Remember the micrometers are expensive, and one can- 
not afford to break them. As suggested in § 74, focus on the edge of 
the cement ring enclosing the lines, then in focusing down to find 
the lines, move the preparation very slightly, back and forth. 

After the lines are sharply focused, and the slide clamped in posi- 
tion make the tube of the microscope horizontal, by bending the flexible 
pillar, being careful not to bring any strain upon the fine adjustment 
(frontispiece). 




CH. IV] 



MAGNIFICATION AND MICROMETRY 



107 



Fig. 97. IVollaslon's Camera Lu- 
cida, showing the rays from the micro- 
scope and from the drawing surface, 
also the position of the pupil of the eye. 

Axis, Axis. Axial rays from 
the microscope and from the drawing 
surface (Ch. V). 

Camera Lucida. A section of the 
quadrangular prism showing the 
course of the rays in the prism from 
the microscope to the eye. As the rays 
are twice reflected, they have the same 
relation on entering the eye that they 
would have by looking directly into the 
ocular. 

A. B. The lateral rays from the 
microscope and their projection upon 
the drawing surface. 

C. D Rays '.from the drawing 
surface to ths eye. 

A. D. A' D' . Overlapping portions of the two fields, where both the micro- 
scopic image and the drawing surface, pencil, etc., can be seen. It is represented 
by the shaded part of the overlapping circles at the right. 

Ocular. The ocular of the microscope. 

P. The drawing pencil. Its point is shown in the overlapping; fields. 




Fig. 97. 



Put a Wollaston camera lucida (Fig. 97 and Ch. V) in position, 
and turn the ocular around if necessary so that the broad flat surface 
may face directly upward, as shown in Fig. 97. Elevate the micro- 
scope by putting a block under the base, so that the perpendicular dis- 
tance from the upper surface of the camera lucida to the table is 250 
mm. (§ 162). Place some white paper on the work-table beneath the 
camera lucida. 

Close one eye, and hold the head so that the other may be very 
close to the camera lucida. Look directly down. The image will ap- 
pear to be on the table. It may be necessary to readjust the focus 
after the camera lucida is in position. If there is difficulty in seeing 
dividers and image consult Ch. V. Measure the image with dividers 
and obtain the power exactly as above (§156-157). 

Thus : Suppose two of the T Vth mm., spaces were taken as object, 
and the image is measured by the dividers, and the spread of the 
dividers is found on the steel rule to be 9f millimeters. If now the 
object is y-fjths of a millimeter and the magnified image is 9A milli- 
meters, the magnification (which is the ratio between size of object 



io8 



MAGNIFICATION AND MICROMETRY 



[CH. IV 



and image) must be gi -r- T 2 T = 47. That is, the magnification is 47 
diameters, or 47 times linear. If the fractional numbers in the above 
example trouble the student, both may be reduced to the same denom- 
ination, thus : If the size of the image is found to be 9A mm. this 
number may be reduced to tenths mm., so it will be of the same 



Image 




Objective 



'^| Object-b 

Object-a 
Fig. 99. 

Figs, 98-99. Figures showing that the size of object and image very directly 
as their distance from the center of the lens. In Fig. gg one can also see why it is 
necessary to focus down, i. e., bring the object and objective nearer together when 
the tube is lengthened. See also fig. 58. 

denomination as the object. In 9 mm. there are 90 tenths, and in f 
there are 4 tenths, then the whole length of the image is 90 -f- 4 = 94 
tenths of a millimeter. The object is 2 tenths of a millimeter, then 



CH. IV] MAGNIFICATION AND MICROMETRY 109 

there must have been a magnification of 94 -r- 2 = 47 diameters in 
order to produce an image 94 tenths of a millimeter long. 

Put the 25 mm. (1 in., C, or X 12) ocular in place of one of 37 
mm. focus, and then put the camera lucida in position. Measure the 
size of the image with dividers and a rule as before. The power will 
be considerably greater than when the low ocular was used. This is 
because the virtual image (Fig. 21, B 3 A 3 ) seen with the high ocular is 
larger than the one seen with the low one. The real image (Fig. 21, 
A 1 ]} 1 ) remains nearly the same, and would be just the same if positive, 
par-focal oculars (§ 37, 72, note) were used. 

Lengthen the tube of the microscope 50-60 mm. by pulling out 
the draw-tube. Remove the camera lucida, and focus, then replace 
the camera and obtain the magnification. It will be greater than with 
the shorter tube. This is because the real image (Fig. 99) is formed 
farther from the objective when the tube is lengthened, and the 
objective must be brought nearer the object. The law is : The size 
of object and image varies directly as their distance from the center of the 
lens. The truth of this statement is illustrated by Figs. 98 and 99. 

§ 161. Varying the Magnification of a Compound Micro- 
scope. — It will be seen from the above experiments (§ 160) that in- 
dependently of the distance at which the microscopic image is 
measured (§ 162), there are three ways of varying the power of a 
compound microscope. These are named below in the order of 
desirability. 

( 1 ) By using a higher or lower objective. 

(2) By using a higher or lower ocular. 

(3) By lengthening or shortening the tube of the microscope (Fig. 
99).* 

§ 162. Standard Distance of 250 Millimeters at which the 
Virtual Image is Measured. — For obtaining the magnification of 
both the simple and the compound microscope the directions were to 
measure the virtual image at a distance of 250 millimeters. This is not 



*Amplifier. — In addition to the methods of varying the magnification given 
in § 161, the magnification is sometimes increased by the use of an amplifier, that 
is a diverging lens or combination placed between the objective and ocular and 
serving to give the image-forming rays from the objective an increased divergence. 
An effective form of this accessory was made by Tolles, who made it as a small 
achromatic concavo-convex lens to be screwed into the lower end of the draw-tube 
(frontispiece) and thus but a short distance above the objective. The divergence 
given to the rays increases the size of the real image about two-fold. 



no 



MAGNIFICATION AND MICROMETRY 



[CH. IV 



sfe 




H 


4 




\\ L ^I — 


— h 




\ 






/ 1 • a 


A 






V ° 
i 


,\ 







Fig. ioo. Figure showing 
the position of the microscope, 
the camera lucida, the eye, and 
the difference in size of the im- 
age depending upon the dis- 
tance at which it is projected 
from the eye. (a) The size at 
25 cm.; (b) at 35 cm., ($ 162). 



that the image could not be seen and measured at any other distance, but 
because some standard must be selected, and this is the most common 
one. The necessity for the adoption of some common standard will be 
seen at a glance in Fig. 100, where is represented graphically the fact 




Fig. 10 i. Sectional view 
of the Abbe Camera Lucida 
to show that in measuring 
the standard distance of 250 
millimeters, one must meas- 
ure along the axis from the 
point P, at the left of the 
prism, to the mirror, and 
from the mirror to the 
drawing surface. For a 
full explanation of this 
camera lucida, see next 
chapter. 



Fig. ioi. 

that the size of the virtual image depends directly on the distance at 
which it is projected, and this size is directly proportional to the ver- 
tical distance from the apex of the triangle, of which it forms a base. 
The distance of 250 millimeters has been chosen on the supposition 
that it is the distance of most distinct vision for the normal human eye. 



CH. IV] 



MAGNIFICATION AND MICROMETRY 



Demonstrate the difference in magnification due to the distance at 
which the image is projected, by raising the microscope so that the 
distance will be 350 millimeters, then lowering to 150 millimeters. 

In preparing drawings it is often of great convenience to make 
them at a distance somewhat less or somewhat greater than the stand- 
ard. In such a case the magnification must be determined for the 
special distance. (See the next chapter, § 181.) 

For discussion of the magnification of the microscope, see : Beale, 
pp. 41, 355 ; Carpenter-Dallinger, p. 288 ; Nageli and Schwendener, 
p. 176 ; Ranvier, p. 29; Robin, p. 126 ; Amer. Soc. Micrs., 1884, p. 
183 ; 1889, p. 22 ; Amer. Jour. Arts and Sciences, 1890, p. 50 ; Jour. 
Roy. Micr. Soc, 1888, 1889. 



■ 


OCULAR 

37 or 5° mm - 


OCULAR 

25 mm. 








Objective. 


TUBE 
IN 


Tube 1 '»•„„„ 

MM. 1 


Tube 
out 

MM. 


Ocular Micrometer 

Valuation, 
tube in. out mm. 




X 


X 


X 


X 








X 


— 

X 


X 


x 








X 


X 


X 


X 








X 


X 


— 

X 


X 








X 


x 


x 


X 








X 


X 


X 


X 






Simple Microscope. 


X 





16- 



FiG. 102. 
Table of Magnifications and of the Valuations of the 



Ocular Micrometer. — The above table should be filled out by each 
student. In using- it for Micrometry and Drawing it is necessary to keep 
clearly in mind the exact conditions under which the determinations were 



1 1 2 MA GNIFICA TION AND MICRO ME TR Y [CH. IV 

-made, a?id also the ways in which variations in magni/icatio?i and the val- 
uation of the ocular micrometer may be produced (§ 161. 162, 172, 176). 

MICROMETRY 

§ 164. Micrometry is the determination of the size of objects by 
the aid of a microscope. 

MICROMETRY WITH THE SIMPLE MICROSCOPE 

§ 165. With a simple microscope (A), the easiest and best way 
is to use dividers and then with the simple microscope determine 
when the points of the dividers exactly include the object. The spread 
of the dividers is then obtained as above (§ 157). This amount will 
be the actual size of the object, as the microscope was only used in 
helping to see when the divider points exactly enclosed the object, and 
then for reading the divisions on the rule in getting the spread of the 
dividers. 

(B) One may put the object under the simple microscope and 
then, as in determining the power (§ 156), measure the image at the 
standard distance. If the size of the image so measured is divided 
by the magnification of the simple microscope, the quotient will give 
the actual size of the object. 

Use a fly's wing or some other object of about that size, and try 
to determine the width in the two ways described above. If all the 
work is accurately done the results will agree. 

MICROxMETRY WITH THE COMPOUND MICROSCOPE 

There are several ways of varying excellence for obtaining the size 
of objects with the compound microscope, the method with the ocular 
micrometer (§ 175-176) being most accurate. 

§ 166. Unit of Measure in Micrometry. — As most of the ob- 
jects measured with the compound microscope are smaller than any of 
the originally named divisions of the meter, and the common or decimal 
fractions necessary to express the size are liable to be unnecessarily 
cumbersome, Harting, in his work on the microscope (1859), proposed 
the one thousandth of a millimeter ( 10 p mm. or 0.001 mm.) or 
one millionth of a meter (to"ooooo or 0.000001 meter) as the unit. He 
named this unit micro-millimeter and designated it mmm. In 1869, 
Listing (Carl's Repetorium fiir Experimental- Physik, Bd, X, p. 5) 



CH. IV] MA GN1FICA TION AND MICROME TRY 113 

favored the thousandth of a millimeter as unit and introduced the name 
Mikron or micrum. In English it is most often written Micron (plural 
micra or microns, pronunciation Mik'ron or Mik'ron). By universal con- 
sent the sign or abbreviation used to designate it is the Greek /x. 
Adopting this unit and sign, one would express five thousandths of a 
millimeter ( 1 5 Q or 0.005 mm.) thus, 5//.* 

\ 167. Micrometry by the use of a stage micrometer on which to mount the ob- 
ject. — In this method the object is mounted on a micrometer and then put under 
the microscope, and the number of spaces covered by the object is read off directly. 
It is exactly like putting any large object on a rule and seeing how many spaces of 
the rule it covers. The defect in the method is that it is impossible to properly 
arrange objects on the micrometer. Unless the objects are circular in outline they 
are liable to be oblique in position, and in every case the end or edges of the object 
may be in the middle of a space instead of against one of the lines, consequently 
the size must be estimated or guessed at rather than really measured. 

§168. Micrometry by dividing the size of the image by the magnifica- 
tion of the microscope. — For example, employ the 3 mm. (}£ in.) objective, 
25 mm.(i in.) ocular, and a Necturus' red blood-corpuscle preparation 
as object. Obtain the size of the image of the long and short axes 
of three corpuscles with the camera lucida and dividers, exactly as in 
obtaining the magnification of the microscope (§ 160). Divide the size 
of the image in each case by the magnification, and the result will be 
the actual size of the blood-corpuscles. Thus, suppose the image of the 
long axis of the corpuscle is 18 mm. and the magnification of the micro- 
scope 400 diameters (§ 154), then the actual length of this long axis of 
the corpuscle is 18 mm.-r- 400=0.045 mm. or 45// (§ 166). 



Fig. 103. Preparation of blood with a 
ring around a group of blood corpuscles. 



As the same three blood-corpuscles are to be measured in three 
ways, it is an advantage to put a delicate ring around a group of three 
or more corpuscles, and make a sketch of the whole enclosed group, 

*The term micromillimeter, abbreviation mmm., is very cumbersome, and be- 
sides is entirely inappropriate since the adoption of the definite meanings for the 
prefixes micro and mega, meaning respectively one-millionth and one million 
times the unit before which it is placed. A micromillimeter would then mean 
one- millionth of a millimeter, not one-thousandth. The term micron has been 
adopted by the great microscopical societies, the international commission on 
weights and measures, and by original investigators, and is, in the opinion of the 
writer, the best term to employ. Jour. Roy. Micr. Soc, 1888, p. 502 ; Nature, 
Vol. XXXVII (1888 J, p. 388. 




H4 MAGNIFICATION AND MICROMETRY \_CH. IV 

marking on the sketch the corpuscles measured (Figs. 61-66). The 
different corpuscles vary considerably in size, so that accurate com- 
parison of different methods of measurement can only be made when 
the same corpuscles are measured in each of the ways. 

§ 169. Micrometry by the use of a Stage Micrometer' and a 
Camera Lucida. — Employ the same object, objective and ocular as 
before. Put the camera lucida in position, and with a lead pencil 
make dots on the paper at the limits of the image of the blood- 
corpuscle. Measure the same three that were measured in § 168. 

Remove the object, place the stage micrometer under the micro- 
scope, focus well, and draw the lines of the stage micrometer so as to 
include the dots representing the limits of the part of the image to be 
measured. As the value of the spaces on the stage micrometer is 
known, the size of the object is determined by the number of spaces 
of the micrometer required to include it. 

This simply enables one to put the image of a fine rule on the 
image of a microscopic object. It is theoretically an excellent method, 
and nearly the same as measuring the spread of the dividers with a 
simple microscope (§ 157, 176). 

OCULAR MICROMETER 

§ 170. Ocular Micrometer, Eye-Piece Micrometer. — This, 
as the name implies, is a micrometer to be used with the ocular. It is 
a micrometer on glass, and the lines are sufficiently coarse to be clearly 
seen by the ocular. The lines should be equidistant and about y-5-th or 
Y^th mm. apart, and every fifth line should be longer and heavier to 
facilitate counting. If the micrometer is ruled in squares {net micro- 
meter) it will be very convenient for many purposes. 

Xhe ocular micrometer is placed in the ocular, no matter what the 
form of the ocular (z. e., whether positive or negative) at the level at 
which the real image is formed by the objective, and the image appears 
to be immediately upon or under the ocular micrometer, and hence 
the number of spaces on the ocular micrometer required to measure 
the real image may be read off directly. This, however, is measuring 
the size of the real image, and the actual size of the object can only be 
determined by determining the ratio between the size of the real image 
and the object. In other words, it is necessary to get the valuation of 
the ocular micrometer in terms of a stage micrometer. 

§ 171. Valuation of the Ocular Micrometer. — This is the 
value of the divisions of the ocular micrometer for the purposes of 



CH. IV] 



MAGNIFICATION AND MICROMETRY 



115 



micrometry, and is entirely relative, depending upon the magnification 
of the real image formed by the objective, consequently it changes 
with every change in the magnification of the real image, and must be 
especially determined for every optical combination (z. e., objective and 
ocular), and for every change in the length of the tube of the micro- 
scope. That is, it is necessary to determine the ocular micrometer val- 
uation for every condition modifying the real image of the microscope 

(§ 161). 

Any Huygenian ocular (Fig. 30) may, however, be used as a micrometer ocu- 
lar by placing the ocular micrometer at the level of the ocular diaphragm, where 
the real image is formed. If there is a slit in the side of the ocular , and the 
ocular micrometer is mounted in some way it may be introduced through the 
opening in the side. When no side opening exists the mounting of the eye-lens 
may be unscrewed and the ocular micrometer, if on a cover-glass can be laid on 
the upper side of the ocular diaphragm. 




Fig. 104. Fig. 105. 

Figs. 104-105. Ocular Micrometer with movable scale. Fig. 104 is a side view 
of the ocular while Fig . 105 gives a sectional end view, and shows the ocular mi- 
crometer hi position . In both the screw which moves the micrometer is shown at 
the left. (Bausch & Lomb Opt. Co.) 

§ 172. Obtaining the Ocular Micrometer Valuation for an 
Ocular Micrometer with Fixed Lines (Figs. 104-105). — Use the 
stage micrometer as object. Light the field well and look into the 
microscope. The lines of the ocular micrometer should be very sharply 
defined. If they are not, raise or lower the eye-lens to make them so ; 
that is, focus as with the simple magnifier. 

When the lines of the ocular micrometer are distinct, focus the 
microscope (§72, 74, 75) for the stage micrometer. The image of the 
stage micrometer will appear to be directly under or upon the ocular 
micrometer. 

Make the lines of the two micrometers parallel by rotating the ocular 
or changing the position of the stage micrometer or both if necessary, 



1 1 6 MA GNIFICA TION AND MICROME TR Y {CH. IV 

and then make any two lines of the stage micrometer coincide with 
any two on the ocular micrometer. To do this it may be necessary to 
pull out the draw- tube a greater or less distance. See how many 
spaces are included in each of the micrometers. 

Divide the value of the included space or spaces on the stage mi- 
crometer by the number of divisions on the ocular micrometer required 
to include them, and the quotient so obtained will give the valuation of 
the .ocular micrometer in fractions of the unit of measure of the stage 
micrometer. For example, suppose the millimeter is taken as the unit 
for the stage micrometer and this unit is divided into spaces of T x v th and 
T -^ ¥ th millimeter. If now, with a given optical combination and tube- 
length, it requires 10 spaces on the ocular micrometer to include the 
real image of T Vth millimeter on the stage micrometer, obviously one 
space on the ocular micrometer would include only one-tenth as much, 
or T Vth mm. h-io = TTnjth mm - That is, each space on the ocular mi- 
crometer would include y^-g-th of a millimeter on the stage micrometer, 
or x^-jjth millimeter of the length of any object under the microscope, 
the conditions remaining the same. Or, in other words, it would re- 
quire ioo spaces on the ocular micrometer to include i millimeter on the 
stage micrometer, then as before, i space of the ocular micrometer would 
have a valuation of y-g-g-th millimeter for the purposes of micrometry. 
The size of any minute object may be determined by multiplying this 
valuation of one space by the number of spaces required to include it. 
For example, suppose the fly's wing or some part of it covered 8 spaces 
on the ocular micrometer, it would be known that the real size of the 
part measured is y-jj-Q-th mm. x 8 = T -§-y mm. or So ju (§ 166). See 
Mark, Jour. Applied Microscopy, Vol. I, p. 4. 

§ 173. Micrometry with the Ocular Micrometer. — Use the 3 
mm. (-|- in. ) objective with the preparation of Necturus blood-corpuscles 
as object. Make certain that the tube of the microscope is of the same 
length as when determining the ocular micrometer valuation. In a 
word, be sure that all the conditions are exactly as when the valuation 
was determined, then put the preparation under the microscope and 
find the same three red corpuscles that were measured in the other 
waj^s (§168-169). 

Count the divisions on the ocular micrometer required to enclose 
or measure the long and the short axis of each of the three corpuscles, 
multiply the number of spaces in both cases by the valuation of the 
ocular micrometer for this objective, tube-length and ocular, and the 
results will represent the actual length of the axes of the corpuscles 
in each case. 



CH. IV] 



MAGNIFICATION AND MICROMETRY 



117 




The same corpuscle is, of course, of the same actual size, when 
measured in each of the three ways, so that if the methods are correct 
and the work carefully enough done, the same results should be ob- 
tained by each method. (§ 176).* 

Fig. 106. Ocular Screw- Micrometer with 
compensation ocular X 6. The upper figure 
shows a sectional view of the ocular and the 
screw for -moving the micrometer at the right. 
At the left is shown a clamping screw to 
fasten the ocular to the upper part of the mi- 
croscope ticbe. Below is a face view, showing 
the graduation on the wheel. An ocular 
piicrometer like this is in general like the 
cob-web micrometer and may be used for 
measuring objects of varying sizes very accu- 
rately. With the ordinary ocular micrometer 
very small objects frequently fill but a part of 
an interval of the micrometer, but with this 
the movable cross lines traverse the object {or 
rather its real image) regardless of the minute- 
ness of the object. {Zeiss' Catalog). 

\ 174. Obtaining the Valuation of the Screw or Filar Micrometer. — This 
micrometer (Fig. 106-107) usually consists of a Ramsden's ocular and cross lines. 
As seen in Fig. 107^ there are three lines. The horizontal and one vertical line 
are fixed. One vertical line may be moved by the screw back and forth across the 
field. 

For obtaining the valuation of this ocular micrometer an accurate stage mi- 
crometer must be used. Carefully focus the xlroth mm. spaces. The lines of the 
ocular micrometer should also be sharp. If the} 7 are not focus them by moving 
the top of the ocular up or down ($ 172). Make the vertical lines of the filar mi- 
crometer parallel with the lines of the stage micrometer. Take the precautions 
regarding the width of the stage micrometer lines given in § 176 (see also Fig. 
108). Note the position of the graduated wheel and of the teeth of the recording 
comb, and then rotate the wheel until the movable line traverses one space on the 
stage micrometer. Each tooth of the recording comb indicates a total revolution 
of the wheel, and by noting the number of teeth required and the graduations on 
the wheel, the revolutions and parts of revolution required to measure the T ^o tn 
mm, of the stage micrometer can be easily noted. Measure in like manner 4 or 5 
spaces and get the average. Suppose this average is iX'th revolutions or 125 
graduations on the wheel, to measure the r tjoth mm. or 10// (see \ 166), then one 
of the graduations on the wheel would measure io/.< divided by 125 =.08/*. In 
using this valuation for actual measurement, the tube of the microscope and the 
objective must be exactly as when obtaining the valuation (see \ 175). 

Example of Measurement. — Suppose one uses the red blood corpuscles of a dog 
or monkey, etc., every condition being as when the valuation was determined, one 
notes very accurately how many of the graduations on the wheel are required to 
make the movable line traverse the object from edge to edge. Suppose it requires 



n8 



MAGNIFICATION AND MICROMETRY 



[CH. IV 



94 of the graduations to measure the diameter, the actual size of the corpuscle 
would be 94X.08 /a = 7.52 u. 

The advantage of the filar micrometer is that the valuation of one graduation 
being so small, even the smallest object to be measured would require several 
graduations to measure it. In ocular micrometers with fixed lines, small objects 
like bacteria might not fill even one space, therefore estimations, not measure- 
ments, must be made. For large objects, like most of the tissue elements, the 
ocular micrometers with fixed lines answer very well, for the part which must be 
estimated is relatively small and the chance of error is correspondingly small. 




Fig. 107. Filar Ocular Micrometer with Field A (Bausch & Lomb, Optical Co.). 

§ 175. Varying the Ocular Micrometer Valuation. — Any 
change in the objective, the ocular or the tube-length of the microscope, 
that is to say, any change in the size of the real image, produces a cor- 
responding change in the ocular micrometer valuation (§ 161, 171, 

176). 

\ 176. Remarks on Micrometry. — In using adjustable objectives (§24, 103), 
the magnification of the objective varies with the position of the adjusting collar, 
being greater when the adjustment is closed as for thick cover-glasses than when 
open, as for thin ones. This variation in the magnification of the objective pro- 
duces a corresponding change in the magnification of the entire microscope, and 
the ocular micrometer valuation — therefore it is necessary to determine the mag- 
nification and ocular micrometer valuation for each position of the adjusting 
collar. 

While the principles of micrometry are simple, it is very difficult to get the 
exact size of microscopic objects. This is due to the lack of perfection and uni- 



*There are three ways of using the ocular micrometer, or of arriving at the size 
of the objects measured with it : 

(A) By finding the value of a division of the ocular micrometer for each optical 
combination and tube-length used, and employing this valuation as a multiplier. 
This is the method given in the text, and ^the one most frequently employed. 
Thus, suppose with a given optical combination and tube-length it required five 
divisions on the ocular micrometer to include the image of f D ths millimeter of the 
stage micrometer, then obviously one space on the ocular micrometer would in- 
clude ith of T 2 oths mm. or 25th mm. ; the size of any unknown object under the 



CH. IV] MAGNIFICATION AND MICROMETRY 119 

formity of micrometers, and the difficulty of determining the exact limits of the 
object to be measured. Hence, all microscopic measurements are only approxi- 
mately correct, the error lessening with the increasing perfection of the apparatus 
and the skill of the observer. 



microscope would be obtained by multiplying the number of divisions on the 
ocular micrometer required to include its image by the value of one space, or in 
this case, ^-th mm. Suppose some object, as the fly's wing, required 15 spaces of 
the ocular micrometer to include some part of it, then the actual size of this part 
of the wing would be 15 X ts = fths, or 0.6 mm. 

(B) By finding the number of divisions on the ocular micrometer required to 
include the image of an entire millimeter of the stage micrometer, and using this 
number as a divisor. This number is also sometimes called the ocular micrometer 
ratio. Taking the same case as in (A), suppose five divisions of the ocular mi- 
crometer are required to include the image of i 2 oths mm., on the stage micrometer, 
then. evidently it would require 5 -s- T \ = 25 divisions on the ocular micrometer to 
include a whole millimeter on the stage micrometer, and the number of divisions 
of the ocular micrometer required to measure an object divided by 25 would give 
the actual size of the object in millimeters or in a fraction of a millimeter. Thus, 
suppose it required 15 divisions of the ocular micrometer to include the image of 
some part of the fly's wing, the actual size of the part included would be 15 -r- 25 
= f or 0.6 mm. This method is really exactly like the one in (A), for dividing by 
25 is the same as multiplying by ^th. 

(C) By having the ocular micrometer ruled in millimeters and divisions of a 
millimeter, and then getting the size of the real image in millimeters. In employ- 
ing this method a stage micrometer is used as object and the size of the image of 
one or more divisions is measured by the ocular micrometer, thus : Suppose the 
stage micrometer is ruled in ^th andy^th mm. and the ocular micrometer is ruled 
in millimeters and T Vth mm. Taking ^ ff th mm. on the stage micrometer as object, 
as in the other cases, suppose it requires 10 of the T \>th mm. spaces or 1 mm. to 
measure the real image, then the real image must be magnified t§-5-t 2 o = 5 diame- 
ters, that is, the real image is five times as great in length as the object, and the 
size of an object may be determined by putting it under the microscope and getting 
the size of the real image in millimeters with the ocular micrometer and dividing 
it by the magnification of the real image, which in this case is 5 diameters. 

Use the fly's wing as object, as in the other cases, and measure the image of 
the same part. Suppose that it required 30 of the ^ mm. divisions = fij mm. or 3 
mm. to include the image of the part measured, then evidently the actual size of 
the part measured is 3 mm, -=- 5 =•§ mm., the same result as in the other cases. 

In comparing these methods it will be seen that in the first two (A and B) the 
ocular micrometer may be simply ruled with equidistant lines without regard to 
the absolute size in millimeters or inches of the spaces. In the last method the 
ocular micrometer must have its spaces some known division of a millimeter or 
inch. In the first two methods only one standard of measure is required, viz, the 
stage micrometer ; in the last method two standards must be used, — a stage mi- 
crometer and an ocular micrometer. Of course, the ocular micrometer in the first 
two cases must have the lines equidistant as well as in the last case, but ruling 
lines equidistant is quite a different matter from getting them an exact division of 
a millimeter or of an inch apart. 



MAGNIFICATION AND MICROMETRY 



\CH. IV 



A difficulty when one is using high powers is the width of the lines of the 
micrometer. If the micrometer is perfectly accurate half the width of each line 
belongs to the contiguous spaces, hence one should measure the image of the space 
from the centers of the lines bordering the space, or as this is somewhat difficult 
in using the ocular micrometer, one may measure from the inside of one border- 
ing line and from the outside of the other. If the lines are of equal width this is 
as accurate as measuring from the center of the lines. Evidently it would not be 
right to measure from either the inside or the outside of both lines (Fig. 108). 

It is also necessary in micrometry to use an objective of sufficient power to 
enable one to see all the details of an object with great distinctness. The necessity 
of using sufficient amplification in micrometry has been especially remarked upon 
by Richardson, Monthly Micr. Jour., 1S74, 1875, ! Rogers, Proc. Amer. Soc. Micro- 
scopists, 1S82, p. 239; Fwell, North American Pract., 1890, pp. 97, 173. 



A 



B 



Fig. 10S. The appearance of the coarse 
stage micrometer and of the fine ocular mi- 
crometer lines when using a high objective. 

{A). The method of measuring the 
spaces by putting the fine ocular mi- 
crometer lines opposite the center of the 
coarse stage micrometer lines. 

(B). Method of measuring the spaces 
of the stage micrometer by putting one 
line of the ocular micrometer (o. m. ) at the 
inside and one at the outside of the coarse 
stage micrometer lines (s. m.). 



Fig. 108. 



As to the limit of accuracy in micrometry, one who has justly earned the right 
to speak with authority expresses himself as follows: " I assume that o.2fx is the 
limit of precision in microscopic measures beyond which it is impossible to go with 
certainty.' 1 '' W. A. Rogers Proc. Amer. Soc. Micrs., 1S83, p. 198. 

In comparing the methods of micrometry with the compound microscope given 
above ($ 167, 168, 169, 175), the one given in \ 167 is impracticable, that given in 
\ 168 is open to the objection that two standards are required, — the stage microme- 
ter, and the steel rule ; it is open to the further objection that several different 
operations are necessary, each operation adding to the probability of error. Theoret- 
ically the method given in \ 169 is good, but it is open to the very serious objection 
in practice that it requires so man}- operations which are especially liable to intro- 
duce errors. The method that experience has found most safe and expeditious, 
and applicable to all objects, is the method with the ocular micrometer. If the 
valuation of the ocular micrometer has been accurately determined, then the only 
difficulty is in deciding on the exact limits of the objects to be measured and so 
arranging the ocular micrometer that these limits are inclosed by some divisions of 
the micrometer. Where the object is not exactly included by whole spaces on the 
ocular micrometer, the chance of error comes in, in estimating just how far into a 
space the object reaches on the side not in contact with one of the micrometer 



\_CH. IV 



MAGNIFICATION AND MICROMETRY 



121 



lines. If the ocular micrometer has some quite narrow spaces, and others consid- 
erably larger, one can nearly always manage to exactly include the object by some 
two lines. The ocular screw micrometers (Fig. 106-107) obviates this entirely as 
the cross hair or lines traverse the object or its real image, and whether this 
distance be great or small it can be read off on the graduated wheel, and no 
estimation or guess work is necessary. 

For those especially interested in micrometry, as in its relation to medical 
jurisprudence, the following references are recommended. These articles consider 
the problem in a scientific as well as a practical spirit : The papers of Prof. Wm. 
A. Rogers on micrometers and micrometry, in the Amer. Quar. Micr. Jour., Vol. 
I, pp. 97, 208 ; Proceedings Amer. Soc. Microscopists, 1882, 1883, 18S7. Dr. M. 
D. Ewell, Proc. Amer. Soc. Micrs., 1890 ; The Microscope, 1S89, pp. 43-45 ; North 
Amer. Pract., 1890, pp. 97, 173. Dr. J. J. Woodward, Amer. Jour, of the Med. 
Sci., 1S75. M. C. White, Article "Blood-stains," Ref. Hand-Book Med. Sciences, 
18S5. Medico-Legal Journal, Vol. XII. For the change in magnification due to 
a change in the adjustment of adjustable objectives, see Jour. Roy. Micr. Soc, 
18S0, p. 702 ; Amer. Monthly Micr. Jour., 1880, p. 67. Carpenter-Dallinger, p. 270. 

If one consults the medico-legal journals ; the microscopical journals, the 
Index Medicus, and the Index Catalog of the Library of the Surgeon General's 
Office, under Micrometry, Blood, and Jurisprudence, he can get on track of the 
main work which has been and is beine done. 






Dry objectives of 16 mm. (f in.), 4 mm. (\ in.) and homogeneous immersion 
objective 0/2 mm. ( T \ in. ) in their mountings. {Bausch & Lomb Opt. Co.). 



CHAPTER V 



DRAWING WITH THE MICROSCOPE 



APPARATUS AND MATERIAL FOR THIS CHAPTER 

Microscope, Abbe and Wollaston's camera lucidas, drawing board, thumb 
tacks, pencils, paper, and microscope screen, (Fig. 59), microscopic preparations. 

DRAWING MICROSCOPIC OBJECTS 

§ 177. Microscopic objects may be drawn free-hand directly from 
the microscope, but in this way a picture giving only the general ap- 
pearance and relations of parts is obtained. For pictures which shall 
have all the parts of the object in true proportions and relations, it is 
necessary to obtain an exact outline of the image of the object, and to 
locate in this outline all the principal details of structure. It is then 
possible to complete the picture free-hand from the appearance of the 
object under the microscope. The appliance used in obtaining out- 
lines, etc., of the microscope image is known as a camera lucida. 

§ 178. Camera Lucida. — -This is an optical apparatus for en- 
abling one to see objects in greatly different situations, as if in one 
field of vision, and with the same eye. In other words it is an optical 
device for superimposing or combining two fields of view in one eye. 

As applied to the microscope, it causes the magnified virtual im- 
age of the object under the microscope to appear as if projected upon 
the table or drawing board, where it is visible with the drawing paper, 
pencil, dividers, etc., by the same eye, and in the same field of vision. 
The microscopic image appears like a picture on the drawing paper 
(see note to § 181). This is accomplished in two distinct ways : 

(A) By a camera lucida reflecting the rays from the microscope so 
that their direction when they reach the eye coincides with that of the 
rays from the drawing paper, pencil, etc. In some of the camera 
lucidas from this group (Wollaston's, Fig. 112), the rays are reflected 
twice, and the image appears as when looking directly into the micro- 
scope. In others the rays are reflected but once, and the image has 
the inversion produced by a plane mirror. For drawing purposes this 



CH. V] 



DRA WING WITH THE MICROSCOPE 



123 



inversion is a great objection, as it is necessary to similarly invert all 
the details added free-hand. 

(B) By a camera lucida reflecting the rays of light from the draw- 
ing paper, etc. , so that their direction when they reach the eye coin- 
cides with the direction of the rays from the microscope (Fig. 58, 109). 
In all of the camera lucidas of this group, the rays from the paper are 
twice reflected and no inversion appears. 





Fig. 109. 



Fig. hi. 

Fig. 109. Abbe Camera Lucida with 
the mirror at 45 , the drawing surface 
horizontal, and the microscope vertical. 

Axis, Axis. Axial ray from the mi- 
croscope and from the drawing surface. 
A, B. Marginal rays of the field on the 
drawing surface, ab. Sectional view of 
the silvered surface on the upper of the tri- 
angular prisms composing the cubical 
prism ( P) . The silvered surface is shown 
as incomplete in the center, thus giving passage to the rays from the microscope. 
Foot. Foot or base of the microscope. 

G. Smoked glass seen in section. It is placed between the mirror and the 
prism to reduce the light from the drawing surface. 

Mirror. The mirror of the camera lucida. A quadrant (Q) has been added 
to indicate the angle of inclination of the mirror, which i?i this case is 45°. 

Ocular. Ocular of the microscope over which the prism of the camera lucida 
is placed. 

F, P. Drawing pencil and the cubical prism over the ocular. 
Fig. 1 10. Geometrical figure showing the angles made by the axial ray with 
the drawing stirface and the mirror. 
A, B. The drawing surface. 

Fig. hi. Ocular showing eye-point, E. P. It is at this point both horizontally 
and vertically that the hole in the silvered surface should be placed (| 182). 



124 DRAWING WITH THE MICROSCOPE [CH V 

The better forms of camera lucidas (Wollaston's, Grunow's, Abbe's, 

etc. ) , may be used for drawing both with low and with high powers. 

Some require the microscope to be inclined (Fig. too) while others are 

designed to be used on the microscope in a vertical position. As in 

biological work, it is often necessary to have the microscope vertical, 

the form for a vertical microscope is to be preferred ; but see Figs. 115- 

116. 

§ 179. Avoidance of Distortion. — In order that the picture 

drawn by the aid of a camera lucida may not be distorted, it is necessary 

that the axial ray from the image on the drawing surface shall be at 7'ight 

angles to the d?-awing sznface (Figs. 112, 114.) 

§ 180. Wollaston's Camera Lucida. — This is a quadrangular prism of glass 
put in the path of the rays from the microscope, and it serves to change the 
direction of the axial ray 90 degrees. In using it the microscope is made horizon- 
tal, and the rays from the microscope enter one-half of the pupil while rays from 
the drawing surface enter the other half of the pupil. As seen in the figure (Fig. 
112), the fields partlj- overlap, and where they do so overlap, pencil or dividers 
and microscopic image can be seen together. 

In drawing or using the dividers with the Wollaston camera lucida it is neces- 
sary to have the field of the microscope and the drawing surface about equally 
lighted. If the drawing surface is too brilliantly lighted the pencil or dividers 
may be seen very clearly, but the microscopic image will be obscure. On the 
other hand, if the field of the microscope has too much light the microscopic 
image will be very definite, but the pencil or dividers will not be visible. It is 
necessary, as with the Abbe camera lucida {\ 182), to have the Wollaston prism 
properly arranged with reference to the axis of the microscope and the eye-point. 
If it is not, one will be unable to see the image well, and may be entirely unable 
to see the pencil and the image at the same time. Again, as rays from the micro- 
scope and from the drawing surface must enter independent parts of the pupil of 
the same eye, one must hold the eye so that the pupil is partly over the camera 
lucida and partly over the drawing surface. One can tell the proper position by 
trial. This is not a very satisfactory camera to draw with, but it is a very good 
form to measure the vertical distance of 250 mm. at which the drawing surface 
should be placed when determining magnification (\ 162). 

§ 181. *Abbe Camera Lucida. — This consists of a cube of glass 
cut into two triangular prisms and silvered on the cut surface of the 



*For some persons the image and drawing surface, pencil, etc., do not appear 
on the drawing board as stated above, but under the microscope, according to the 
general principle that "objects appear in space where they could be touched 
along a perpendicular to the retinal surface stimulated," — that is in the line of 
rays entering the eye. This is always the case with the Wollaston camera lucida. 
The explanation of the apparent location of the image, etc., on the drawing board 
with the Abbe camera lucida is that the attention is concentrated upon the draw- 
ing surface rather than upon the object under the microscope (Dr. W. B. 
Pillsbury). 



CH V] 



DRA WING WITH THE MICROSCOPE 



125 



upper one. A small oval hole is then cut out of the center of the sil- 
vered surface and the two prisms are cemented together in the form of 
the original cube with a perforated 45 degree mirror within it (Fig. 
109, ab). The upper surface of the cube is covered by a perforated 
metal plate. This cube is placed over the ocular in such a way that 
the light from the microscope passes through the hole in the silvered 
face and thence directly to the eye. Light from the drawing surface 
is reflected by a mirror to the silvered surface of the prism and reflect- 
ed by this surface to the eye in 
company with the rays from the 
microscope, so that the two fields 
appear as one, and the image is 
seen as if on the drawing surface 
(Figs. 109, 114). It is designed 
for use with a vertical micro- 
scope, but see § 184. 

Fig. 112. Wollaslon's Camera 
Lucida, showing the rays from the 
microscope and from the drawing sttr- 
face, and the position of the pupil of 
the eye. 

For full explanation see Fig. 97. 




§ 182. Arrangement of the Camera Lucida Prism. — In plac- 
ing this camera lucida over the ocular for drawing or the determination 
of magnification, the center of the hole in the silvered surface is placed 
in the optic axis of the microscope. This is done by properly arrang- 
ing the centering screws that clamp the camera to the microscope tube 
or ocular. The perforation in the silvered surface must also be at the 
level of the eye-point. In other words the prism must be so arranged 
vertically and horizontally that the hole in the silvered surface will be 
in the axis of the microscope and coincident with the eye-point of 
the ocular. If it is above or below, or to one side of the eye-point, 
part or all of the field of the microscope will be cut off. As stated 
above, the centering screws are for the proper horizontal arrangement 
of the prism. The prism is set at the right height by the makers for 
the eye-point of a medium ocular. If one .desires to use an ocular 
with the eye-point farther away or nearer, as in using, high o\ low 
oculars the position of the eye-point may be determined as directd in 



126 



DRAWING WITH THE MICROSCOPE 



[CH V 




Fig. 113. One of the latest and 
best forms of the Abbe Camera Lucid a 
{Bausch & Lomb Optical Co. ). 



§ 59 and the prism loosened and raised or lowered to the proper level ; 
but in doing this one should avoid setting the prism obliquely to the 
mirror. 

In the latest and best forms of this camera lucida special arrange- 
ments have been made for raising or lowering the prism so that it may 
be used with equal satisfaction on oculars with the eye-point at differ- 
ent levels, and the prism is hinged to turn aside without disturbing 
the mirror. 

One can determine when the 
camera is in a proper position by 
looking into the microscope through 
it. If the field of the microscope 
appears as a circle and of about the 
same size as without the camera 
lucida, then the prism is in a proper 
position. If one side of the field is 
dark, then the prism is to one side 
of the center ; if the field is consid- 
erably smaller than when the prism is turned off the ocular, it indicates 
that it is not at the correct level, i. e., it is above or below the eye- 
point. 

§ 183. Arrangement of the Mirror and the Drawing Surface. 
—The Abbe camera lucida was designed for use with a vertical micro- 
scope (Fig. 109). On a vertical microscope if the mirror is set at an 
angle of 45 , the axial ra3 T will be at right angles with the table top or a 
drawing board which is horizontal, and a drawing made under these 
conditions will be in true proportion and not distorted. The stage of 
most microscopes, however, extends out so far at the sides that with 
a 45 mirror the image appears in part on the stage of the microscope. 
In order to avoid this the mirror may be depressed to some point below 
45 , say at 40 or 35°(Fig. 1 14). But as the axial ray from the mirror 
to the prism must still be reflected horizontally, it follows that the axial 
ray will no longer form an angle of 90 degrees with the drawing sur- 
face, but a greater angle. If the mirror is depressed to 35 , then the 
axial ray must take an angle of no° with a horizontal drawing surface 
(see the geometrical figure Fig. 114, A). To make the angle 90 again, 
so that there shall be no distortion, the drawing board must be raised 
toward the microscope 20 . The general rule is to raise the draw- 
ing board twice as many degrees toward the microscope as the 
mirror is depressed below 45 °. Practically the field for drawing 



CH. V\ 



DRA WING WITH THE MICROSCOPE 
B 



127 




Fig. 114. C 

Abbe Camera Lucida in position to avoid distortion. 

Fig. 114. — The Abbe Camera Lucida with the mirror at 35°. 

Axis, Axis. Axial ray from the microscope and from the drawing surface. 

A. B. Drawing surface raised toward the microscope 20° . 

Foot. The foot or base of the microscope. 

Mirror with quadrant {Q). The mirror is seen to be at an angle of 35° . 

Ocular. Ocular of the Microscope. 

P. P. Drawing pencil and the cubical prism over the ocular. 

W. Wedge to support the drawing board. 

A. Geometrical figure of the preceding, showing the angles made by the 
axial ray with the mirror and the necessary elevation of the drawing board to 
avoid distortion. From the equality of opposite angles, the angle of the axial ray 
reflected at 35° must make an angle of 110° with a horizontal drawing board. The 
board must then be elevated toward the microscope 20° in order that the axial ray 
may be perpendicular to it, and thus fulfill the requirements necessary to avoid 
distortion (■§ ijg, 1S3). 

B. Upper view of the prism of the camera lucida. A considerable portion of 
the face of the prism is covered, and the opening in the silvered surface appears 
oval. 

C. Quadrant to be attached to the mirror of the Abbe Camera Lucida to in- 
dicate the angle of the mirror. As the angle is nearly always 43 , 40°, or 35°, 
only those angles are shown. 



128 DRAWING WITH THE MICROSCOPE [CH. V 

can always be made free of the stage of the microscope, at 45 , at 40 , 
or at 35 . In the first case (45 mirror) the drawing surface should 
be horizontal, in the second case (40 mirror) the drawing surface 
should be elevated io°, and in the third case (35 mirror) the drawing 
board should be elevated 20 toward the microscope. Furthermore it 
is necessary in using an elevated drawing board to have the mirror bar 
project directly laterally so that the edges of the mirror will be in 
planes parallel with the edges of the drawing board, otherwise there 
will be front to back distortion, although the elevation of the drawing 
board would avoid right to left distortion. If one has a micrometer 
ruled in squares {net micrometer) the distortion produced by not having 
the axial ray at right angles with the drawing surface may be very 
strikingly shown. For example, set the mirror at 35 and use a hori- 
zontal drawing board. With a pencil make dots at the corners of 
some of the squares, and then with a straight edge connect the dots. 
The figures will be considerably longer from right to left than from 
front to back. Circles in the object would appear as ellipses in the 
drawings, the major axis being from right to left. 

The angle of the mirror may be determined with a protractor, but 
that is troublesome. It is much more satisfactor}' to have a quadrant 
attached to the mirror and an indicator on the projecting arm of the 
mirror. If the quadrant is graduated throughout its entire extent, or 
preferably at three points, 45 °, 40 and 35 °, one can set the mirror at a 
known angle in a moment, then the drawing board can be hinged and 
the elevation of io° and 20 determined with a protractor. The draw- 
ing board is very conveniently held up by a broad wedge. By marking 
the position of the wedge for io° and 20 the protractor need be used 
but once, then the wedge may be put into position at any time for the 
proper elevation. 

§ 184. Abbe Camera and Inclined Microscope. — It is very 
fatiguing to draw continuously with a vertical microscope, and many 
mounted objects admit of an inclination of the microscope, when one 
can sit and work in a more comfortable position. The Abbe camera is 
as perfectly adapted to use with an inclined as with a vertical micro- 
scope. All that is requisite is to be sure that the fundamental law is 
observed regarding the axial ray of the image and the drawing surface, 
viz., that they should be at right angles. This is very easily accom- 
plished as follows : The drawing board is raised toward the microscope 
twice as many degrees as the mirror is depressed below 45 ° (§ 183;, 
then it is raised exactly as many degrees as the microscope is inclined, 



CH V\ 



DRA WING WITH THE MICROSCOPE 



129 




and in the same direction, that is, so the end of the drawing board 

shall be in a plane parallel with the stage of the microscope. The 

mirror must have its edges in planes parallel with the edges of the 
drawing board also (Figs. 115, 116.) 



Fig. 115. Arrangement of 
the drawing board for using the 
microscope in an inclined position 
with the Abbe camera lucida (de- 
signed by Mrs. S. P. Gage, 18S7.) 



§ 1S5. Drawing with the Abbe Camera Lucida. — (A) The 
light from the microscope and from the drawing surface should be of 
nearly equal intensity, so that the image and the drawing pencil can be 
seen with about equal distinctness. This may be accomplished with 
ver} T low powers (16 mm. and lower objectives) by covering the mirror 
of the microscope with white paper when transparent objects are to be 
drawn. For high powers it is best to use a substage condenser. Often 
the light may be balanced by using a larger or smaller opening in the 
diaphragm. One can tell which field is excessively illuminated, for 
it is the one in which objects are most distinctly seen. If it is the 
microscopic, then the image of the microscopic object is very distinct 
and the pencil is invisible or very indistinct. If the drawing surface is 
too brilliantly lighted the pencil can be seen clearly, but the micro- 
scopic image will be obscure. 

When opaque objects, that is objects which must be lighted with 
reflected light (§ 63), like dark colored insects, etc., are to be drawn 
the light must usually be concentrated upon the object in some way. 
The microscope may be placed in a very strong light and the drawing 
board shaded or the light may be concentrated upon the object by 
means of a concave mirror or a bull's eye condenser (Fig. 53). 

If the drawing surface is too brilliantly illuminated, it may be 
shaded by placing a book or a ground glass screen between it and the 
window, also by putting one or more smoked glasses in the path of the 
rays from the mirror (Fig. 109 G). If the light in the microscope is 
too intense, it may be lessened by using white paper over the mirror, 
or by a ground glass screen between the microscope mirror and the 
source of light (Piersol, Amer. M. M. Jour., 1888, p. 103). It is also 
an excellent plan to blacken the end of the drawing pencil with carbon 



130 



DRAWING WITH THE MICROSCOPE 



\CH. V 



ink. Sometimes it is easier to draw on a black surface, using a white 
pencil or style. The carbon paper used in manifolding letters, etc., 
may be used, or ordinary black paper may be lightly rubbed on one 
side with a moderately soft lead pencil. Place the black paper over 
white paper and trace the outlines with a pointed style of ivory or 
bone. A corresponding dark line will appear on the white paper 
beneath. (Jour. Roy. Micr. Soc, 1883, p. 4 2 3)- 




Fig. 116. Bern hard'- 's Drawing Board for the Abbe Camera Lucida. This 
drawing board is adjustable vertically, and the board may be inclined to prevent 
distortion. It is also arranged for use with an inclined microscope , having the 
base board hinged. ' Microscope and drawing surface are then inclined together. 
{Zeit. wiss. Mikroskopie, vol. vi (1894, p. 29S). {Zeiss Catalog). 

(A) It is desirable to have the drawing paper fastened with thumb 
tacks, or in some other way. (B) The lines made while using the 
camera lucida should be very light, as they are liable to be irregular. 
(C) Only outlines are drawn and parts located with a camera lucida. 
Details are put in free-hand. (D) It is sometimes desirable to draw 
the outline of an object with a moderate power and add the details with 
a higher power. If this is done it should always be clearly stated. It 
is advisable to do this only with objects in which the same structure is 
many times duplicated, as a nerve or a muscle. In such an object all 



CH. V~\ DRAWING WITH THE MICROSCOPE 131 

the different structures could be shown, and by omitting some of the 
fibers the others could be made plainer without an undesirable enlarge- 
ment of the entire figure. 

(E) If a drawing of a given size is desired and it cannot be ob- 
tained by any combination of oculars, objectives and lengths of the 
tube of the microscope, the distance between the camera lucida and 
the table may be increased or diminished until the image is of the 
desired size. This distance is easily changed by the use of a book or 
a block, but more conveniently if one has a drawing board with adjust- 
able drawing surface like that shown in Fig. 116. The image of a few 
spaces of the micrometer will give scale of enlargement, or the power 
ma}- be determined for the special case (§ 186-187). 

(F) It is of the greatest advantage, as suggested by Heinsius (Zeit. 
w. Mikr., 1889, p. 367), to have the camera lucida hinged so that the 
prism may be turned off the ocular for a moment's glance at the prepa- 
ration, and then returned in place without the necessity of loosening 
screws and readjusting the camera. This form is now made by several 
opticians, and the quadrant is added hy some. (Fig. 114.) Any skilled 
mechanic can add the quadrant. 

§ 186. Magnification of the Microscope and size of Draw- 
ings with the Abbe Camera Lucida. — In determining the standard 
distance of 250 millimeters at which to measure the image in getting 
the magnification of the microscope, it is necessary to measure from 
the point marked P on the prism (Fig. 109) to the axis of the mirror 
and then vertically to the drawing board. 

In getting the scale to wdiich a drawing is enlarged the best way 
is to remove the preparation and put in its place a stage micrometer, 
and to trace a few (5 or 10) of its lines upon one corner of the drawing. 
The value of the spaces of the micrometer being given, thus : 



,fotli mm. 

Fig. 117. Showing the method of indicating the scale at which a drawing 
was made. 

The enlargement of the figure can then be accurately determined 
at any time by measuring with a steel scale the length of the image of 
the micrometer spaces and dividing it by their known size. 

Thus, suppose the 5 spaces of the scale of enlargement given with 
a drawing were found to measure 25 millimeters and the spaces on the 



132 



DRA WING WITH THE MICROSCOPE 



[CH. V 



micrometer were 

25 -5" t4 = 500 



y 1 th millimeter, then the enlargement would be 
That is, the image was drawn at a magnification of 



100 
500 diameters. 

If the micrometer scale is added to every drawing, there is no 
need of troubling one's self about the exact distance at which the 
drawing is made, convenience may settle that, as the special magnifi- 
cation in each case may be determined from the scale accompanying 
the picture. It should be remembered, however, that the conditions 
when the scale is drawn must be exactly as when the drawing was 
made. 

§ 187. Drawing at Slight Magnification. — Some objects are of 
considerable size and for drawings should be enlarged but a few diame- 
ters, — -5 to 20. By using sufficiently low objectives and different ocu- 
lars a great range may be obtained. Frequently, however, the range 
must be still further increased. For a moderate increase in size the 
drawing surface may be put farther off, or, as one more commonly 
needs less rather than greater magnification, the drawing surface may 
be brought nearer the mirror of the camera lucida by piling books or 
other objects on the drawing board. If one takes the precaution to 
draw a scale on the figure under the same conditions, its enlargement 
can be readily determined (§ 186). 

A very satisfactorj- way to draw at low magnifications is to use a 
simple microscope and arrange a camera lucida over it as over the ocular. 
In this way one may get drawings at almost any low magnification. 

If one has many 
large objects to draw at a 
low magnification, then 
some form of embryo- 
graph is very conven- 
ient. (Jour. Roy. Micr., 
Soc, 1899, p. 223.) The 
writer has made use of 
a photographic camera 
and different photo- 
graphic objectives for the 
purpose. The object is 

Fig. 118. Camera lucida for drawing objects natural size. (H. Bausch 
Jour. Applied Microscopy, vol. Hi {iqoo, p. Sgi). 

illuminated as if for a photograph and in place of the ground glass a 
plain glass is used and on this some tracing paper is stretched. Noth- 




CH. V] 



DRA WING WITH THE MICROSCOPE 



133 



ing is then easier than to trace the outlines of the object. See also Ch. 
VIII. 

REFERENCES 

Beale, 31, 355 ; Behrens, Kossel and Schiefferdecker, 77 ; Carpenter-Dallinger, 
278 ; Van Heurck, 91 ; American Naturalist, 1886, p. 1071, 1887, pp. 1040-1043 ; 
Amer. Monthly Micr. Jour., 1S88, p. 103, 1890, p. 94 ; Jour. Roy. Micr. Soc, 1881, 
p. 819, 18S2, p. 402, 1883, pp. 283, 560, 1884, p. 115, 1886, p. 516, 1888, pp. 113, 809, 
798; Zeit. wiss. Mikroskopie, 1884, pp. 1-21, 1889, p. 367, 1893, pp. 289-295. 
Here is described an excellent apparatus made by Winkel. Consult also the latest 
catalogs of the opticians. 




10 CENTIMETER RULE 

The upper edge is in millimeters, the lower in centimeters, and half centimeters. 



THE METRIC SYSTEM 



UNITS. 



The most commonly used divisions and multiples. 



( Centimeter (cm.), i-iooth Meter ; Millimeter (m.m.j, i-ioooth Meter: Micron 
length I (/*)' i-ioooth Millimeter; the Micron is the unit in Micrometry (% 166). 

(Kilometer, 1000 Meters ; used in measuring roads and other long distances. 

the gram for \ Milligram (m.g. ) , i-ioooth Gram. 
weight. . . ( Kilogram, 1000 Grams, used for ordinary masses, like groceries, etc. 

the liter for j Cubic Centimeter (c.c), i-ioooth Liter. This is more common than the correct 
capacity. . ( form, Milliliter. 

Divisions of the Units are indicated by the Latin prefixes : deci, i-ioth ; centi, i-iooth ; Milli, 
i-ioooth ; Micro, i-i,ooo,oooth of any unit. 

Multiples are designated by Greek prefixes : deka, 10 times ; hecto, 100 times ; kilo, 1000 times 
myria, 10,000 times ; Mega, 1,000,000 times any unit. 



CHAPTER VI 

MICRO-SPECTROSCOPE AND POLARISCOPE, 
MICRO-CHEMISTRY, MICRO-MATALXOGRAPHY, 

TEXTILE FIBERS 



APPARATUS AND MATERIAL REQUIRED FOR THIS CHAPTER 

Compound microscope ; Micro-spectroscope (? iSS) ; Watch-glasses and small 
vials, slides and covers (§ 207) ; Various substances for examination (as blood and 
ammonium sulphide, permanganate of potash, chlorophyll, some colored fruit, 
etc., {I 208-217); Micro-polarizer (? 21S); Selenite plate (? 227); Various doubly 
refracting objects, as crystals, textile fibers, starch, section of bone ; Various 
chemicals, metals, etc. 

MICRO-SPECTROSCOPE 

\ 1S8. A Micro-Spectroscope, Spectroscopic or Spectral Ocular, is a direct 
vision spectroscope in connection with a microscope ocular. The one devised by 
Abbe and made by Zeiss consists of a direct vision spectroscope prism of the Amici 
pattern, and of considerable dispersion, placed over the ocular of the microscope. 
fThis direct vision or Amici prism consists of a single triangular prism of heavy 
flint glass in the middle and one of crown glass on each side, the edge of the 
crown glass prisms pointing toward the base of the flint glass prism, i. e., the edge 
of the crown and flint glass prisms point in opposite directions. The flint glass 
prism serves to give the dispersion or separation into colors, while the crown glass 
prisms serve to make the emergent rays approximately parallel with the incident 
rays, so that one looks directly into the prism along the axis of the microscope^/ 

The Amici prism is in a special tube which is hinged to the ocular and held in 
position by a spring. It may be swung free of the ocular. In connection with 
the ocular is the slit mechanism and a prism for reflecting horizontal rays verti- 
cally for the purpose of obtaining a comparison spectrum (| 201). Finally near 
the top is a lateral tube with mirror for the purpose of projecting an Angstrom 
scale of wave lengths upon the spectrum (g 202, Figs. 1 19-120. ) 

I 189. Apparent Reversal of the Position of the Colors in a Direct Vision 
Spectroscope. — In accordance with the statements in \ 188 the dispersion or sepa- 
ration into colors is given by the flint glass prism or prisms and in accordance 
with the general law that the waves of shortest length, blue, etc., will be bent 
most, the colors have the position indicated in the top of Fig. 123, also above 
Fig. 119. But if one looks into the direct vision spectroscope or holds the e3 - e 
close to the single prism (Fig. 124), the colors will appear reversed as if the red 
were more bent. The explanation of this is shown in Fig. 124, where it can be 



CH. VI} MICRO-SPECTROSCOPE AND POLARISCOPE 



35 



readily seen that if the eye is placed at E, close to the prism, the different colored 
rays appear in the direction from which they reach the eye and consequently 
are crossed in being projected into the field of vision and the real position is in- 
verted. The same is true in looking into the micro-spectroscope. The actual 





4 

Fig. 119. Abbe's Micro-spectroscope. Fig. 120. 

Longitudinal Section of Slit Mechanism separately . 

the whole instrument. {Plan view, Full size. ) 

( % Full size. ) 

" The eye lens is adjustable so as to accurately focus on the slit situated between 
the lenses. The mechanism for contracting and expanding the slit is actuated by 
the screw F and causes the laminae to move symmetrically (Merz's movement). 
The slit may be made sufficiently wide so as to include the whole visual field. The 
screw H serves to limit the length of the slit so as to completely fill the latter with 
the image of the object under investigation when the comparison prism is inserted. 
The comparison prism is provided with a lateral frame and clips to hold the object 
and the illuminating mirror. All these parts are encased in a drum on the ocular. 

Above the eye-piece is placed an Amid prism of great dispersion which may be 
turned aside about the pivot K, so as to allow of the adjustment of the object. The 
prism is retained in its axial position by the spring catch L. A scale is projected 
on the spectrum by means of a scale tube and mirror attached to the prism casing. 
The divisions of the scale indicate in decimals of a micron the wave length of the 
respective section of the spectrum. The screw P serves to adjust the scale relative 
to the spectrum. 

The instrument is inserted in the tube in place of the ordinary eye-piece and is 
clamped to the former by means of the screw M in such a position that the mirrors 
A and O, which respectively serve to illuminate the comparison prism and the 
scale of wave lengths are simultaneously illuminated." (Cut loaned by Wm. 
Krafft, N. Y. ) 



136 



MICRO-SPECTROSCOPE AND POLAR/SCOPE \CH. VI 



position of the different colors may be determined by placing some ground glass 
or some of the lens-paper near the prism and observing with the eye at the 
distance of distinct vision.* 



J 


iuiiii 


) ( 


1 


3 

.40 
I 1 1 1 


) E 

[ I i 1 l 1 i I 


F 

6.0 

1 I i i i 


a 

Ho 

1 , , , , 1 


< 

o 






l! 
it- 












C 














P* 


£o<Uuvt\ 




'J 
















'-ra 






:', 




I'll 1 ! 1 11 I 




I 




'73- 

C5' 


















WUUat-| 




; 


1 ,. 


Jl ,;m 


I'm 


Ek 








p 

4 



Fig. i2i. Various Spectrums.—All except that of sodium were obtained by 
diffused day-light with the slit of such a width as gave the most distinct Fraunhofer 
lines. 

It frequently occurs that with a substance giving several absorption bands (e. 
g., chlorophyll) the density or thickness of the solution must be varied to show all 
the different bands clearly. 

Solar Spectrum.— With diffused day -light and a narrow slit the spectrum is not 
visible much beyond the fixed line B. In order to extend the visible spectriim in 
the red to the line A, one should use direct sunlight and a piece of ruby glass in 
place of the watch-glass in Fig. 123. 

Sodium Spectrum.— The line spectrum {\i 91) of sodium obtained by lighting 
the microscope with an alcohol flame in which some salt of sodium is glowing. 
With the micro-spectroscope the sodium line seen in the solar spectrum and with 
the incandescent sodium appears single, except tinder very favorable circumstances. 
{\ 192). By using a comparison spectrum of day-light with the sodium spectrum 
the light and dark D-lines will be seen to be continuous as here shown. 

Permanganate of Potash. — This spectrum is characterized by the presence of 
five absorption bands in the middle of the spectrum and is best shown by using a T \ 
per cent, solution of permanganate in water in a watch-glass as in Fig. 123. 

Met-hemoglobin . — The absorption spectrum of met-hemoglobin is characterized 
by a considerable darkening of the blue end of the spectrum and of four absorption 
bands, one in the red near the line C and two between D and E nearly in the place 
of the two bands of oxy-hemoglobin ; finally there is a somewhat faint, wide, band 
near F. Such a met-hemoglobin spectrum is best obtained by making a solution of 
blood in water of such a concentration that the two oxy-hemoglobin bands run 
together {I 211), and then adding three or four drops of a t l per cent, aqueous 
solution of permanganate of potash or a few drops of hydrogen dioxid (H 2 0., )• 
Soon the bright red will change to a brownish color, when it may be examined. 

*The author wishes to acknowledge the aid rendered by Professor E. L. 
Nichols in giving the explanation offered in this section. 



CH. VI] MICRO-SPECTROSCOPE AND POLARISCOPE 137 

VARIOUS KINDS OF SPECTRA 

By a spectrum is meant the colored bands appearing when the light traverses 
a dispersing prism or a diffraction grating, or is affected in any way to separate 
the different wave lengths of light into groups. When daylight or some good 
artificial light is thus dispersed one gets the appearance so familiar in the 
rainbow. 

\ 190. Continuous Spectrum. — In case a good artificial light as the electric 
light is used the various rainbow or spectral colors merge gradually into one 
another in passing from end to end of the spectrum. There are no breaks or gaps. 

$ 191. Line Spectrum. — If a gas is made incandescent, the spectrum it pro- 
duces consists, not of the various rainbow colors, but of sharp, narrow, bright lines, 
the color depending on the substance. All the rest of the spectrum is dark. 
These line spectra are very strikingly shown by various metals heated till they are 
in the form of incandescent vapor. 

I 192. Absorption Spectrum. — By this is meant a spectrum in which there are 
dark lines or bands in the spectrum. The most striking and interesting of the 
absorption spectra is the Solar Spectrum, or spectrum of sunlight. If this is exam- 
ined carefully it will be found to be crossed by dark lines, the appearance being as 
if one were to draw pen marks across a continuous spectrum at various levels, 
sometimes apparently between the colors and sometimes in the midst of a color. 
These dark lines are the so-called Fraunhofer Lines. Some of the principal ones 
have been lettered with Roman capitals, A, B, C, D, E, F, G, H, commencing at 
the red end. The meaning of these lines was for a long time enigmatical, but it 
is now known that they correspond with the bright lines of a line spectrum (§ 191). 
For example, if sodium is put in the flame of a spirit lamp it will vaporize and 
become luminous. If this light is examined there will be seen one or two bright 
yellow bands corresponding in position with D of the solar spectrum (Fig. 121). 
If now the spirit-lamp flame, colored by the incandescent sodium, is placed in the 
path of the electric light, and it is examined as before, there will be a continuous 
spectrum, except for dark lines in place of the bright sodium lines. That is, the 
comparatively cool yellow light of the spirit lamp cuts off or absorbs the intensely 
hot yellow light of the electric light ; and although the spirit flame sends a yellow 
light to the spectroscope it is so faint in comparison with the electric light that the 
sodium lines appear dark. It is believed that in the sun's atmosphere there are 
incandescent metal vapors (sodium, iron, etc. ), but that they are so cool in com- 
parison with the rays of their wave length in the sun that the cooler light of the 
incandescent metallic vapors absorb the light of corresponding wave length, and 
are, like the spirit lamp-flame, unable to make up the loss, and therefore the pres- 
ence of the dark lines. 

\ 193. Absorption Spectra from Colored Substances. — While the solar spec- 
trum is an absorption spectrum, the term is more commonly applied to the spectra 
obtained with light which has passed through or has been reflected from colored 
objects which are not self-luminous. 

It is the special purpose of the micro-spectroscope to investigate the spectra of 
colored objects which are not self-luminous, i. e., blood and other liquids, various 
minerals, as monazite, etc. The spectra obtained by examining the light reflected 
from these colored bodies or transmitted through them, possess, like the solar 



133 



MICRO-SPECTROSCOPE AND POLAR/SCOPE \_CH. VI 



spectrum dark lines or bands, but the bands are usually much wider and less 
sharply defined. Their number and position depend on the substance or its con- 
stitution (Fig. 122), and their width, in part, upon the thickness of the body. 
With some colored bodies, no definite bands are present. The spectrum is simply 
restricted at one or both ends and various of the other colors are considerably 
lessened in intensity. This is true of many colored fruits. 

\ 194. Angstrom and Stokes' Law of Absorption Spectra. — The waves of 
light absorbed by a body when light is transmitted through some of its substance 



.90 .80 




A a, 8 c 
70 



60 

nul l 



,1 <0r,*gsfodlo 



1 



.So 

_J_i 



73 



J_ I L 



B L u S 




Fig. 122. Absorption spectrum of Oxy-hemoglobiii or arterial blood (/) and 
of Homoglobin or venous blood (2). (From Gamgee and McMunn). 

A, B, C, D, E, E, G, H. Some of the Prinipal Fraunhofer lines of the solar 
spectrum (\ 192). 

.go, .So, .jo, .60, .50, .40. Wave lengths in microns, as shown in Angstrom'' s 
scale (§ 202). It will be seen that the wave lengths increase toward the red and 
decrease toward the violet end of the spectrum. 

Red, Yellow, Orange, etc. Color regions of the spectrum. Indigo should 
come between the blue and the violet to complete the seven colors usually given. It 
was omitted through inadvertence. 

are precisely the waves radiated from it when it becomes self-luminous. For ex- 
ample, a piece of glass that is yellow when cool, gives out blue light when it is hot 
enough to be self-luminous. Sodium vapor absorbs two bands of yellow light (D 
lines); but when light is not sent through it, but itself is luminous and examined 
as a source of light its spectrum gives bright sodium lines, all the rest of the 
spectrum being dark (Fig. 121). 

I 195. Law of Color. — The light reaching the eye from a colored, solid, 
liquid or gaseous body lighted with white light, will be that due to white light less 
the light waves that have been absorbed by the colored body. Or in other words, 
it will be due to the wave lengths of light that finally reach the eye from the ob- 
ject. For example, a thin layer of blood under the microscope will appear 
yellowish green, but a thick layer will appear pure red. If now these two layers 
are examined with a micro-spectroscope, the thin layer will show all the colors, but 
the red end will be slightly, and the blue end considerably restricted, and some of 
the colors will appear considerably lessened in intensity. Finally there may ap- 
pear two shadow-like bands, or if the layer is thick enough, two well-defined 
dark bands in the green (§ 210). 

If the thick layer is examined in the same way, the spectrum will show only 
red with a little orange light, all the rest being absorbed. Thus the spectroscope 
shows which colors remain, in part or wholly, and it is the mixture of this remain- 
ing or unabsorbed li^ht that gives color to the obiect. 



CH. VI -\ MICRO-SPECTROSCOPE AND POLARISCOPE 139 

I 196. Complementary Spectra. — "While it is believed that Angstrom's law 
{\ 194) is correct, there are many bodies on which it cannot be tested, as they 
change in chemical or molecular constitution before reaching a sufficiently high 
temperature to become luminous. There are compounds, however, like those of 
didymium, erbium and terbium, which do not change with the heat necessary to 
render them luminous, and with them the incandescence and absorption spectra 
are mutually complementary, the one presenting bright lines where the other 
presents dark ones (Daniell). 

ADJUSTING THE MICRO-SPECTROSCOPE 

§ 197. The micro-spectroscope, or spectroscopic ocular, is put in 
the place of the ordinary ocular in the microscope, and clamped to the 
top of the tube by means of a screw for the purpose. 

§ 198. Adjustment of the Slit. — In place of the ordinary dia- 
phragm with circular opening, the spectral ocular has a diaphragm 
composed of two movable knife edges by which a slit-like opening of 
greater or less width and length may be obtained at will by the use of 
screws for the purpose. To adjust the slit, depress the lever holding 
the prism-tube in position over the ocular, and swing the prism aside. 
One can then look into the ocular. The lateral screw should he used 
and the knife edges approached till they appear about half a millimeter 
apart. If now the Amici prism is put back in place and the micro- 
scope well lighted, one will see a spectrum by looking into the upper 
end of the spectroscope. If the slit is too wide, the colors will overlap 
in the middle of the spectrum and be pure only at the red and blue 
ends ; and the Fraunhofer or other bands in the spectrum will be 
faint or invisible. Dust on the edges of the slit gives the appearance 
of longitudinal streaks on the spectrum. 

§ 199. Mutual Arrangement of Slit and Prism. — In order 
that the spectrum may appear as if made up of colored bands going 
directly across the long axis of the spectrum, the slit must be parallel 
with the refracting edge of the prism. If the slit and prism are not 
thus mutually arranged, the colored bands will appear oblique, and 
the whole spectrum may be greatly narrowed. If the colored bands 
are oblique, grasp the prism tube and slowly rotate it to the right or to 
the left until the various colored bands extend directly across the spec- 
trum. 

§ 200. Focusing the Slit. — In order that the lines or bands in 
the spectrum shall be sharply defined, the eye-lens of the ocular should 
be accurately focused on the slit. The eye-lens is movable, and when 
the prism is swung aside it is very easy to focus the slit as one focused 



140 



MICRO-SPECTROSCOPE AND POLAR/SCOPE [CH. VI 



for the ocular micrometer (§ 172). If one now uses daylight there 
will be seen in the spectrum the dark Fraunhofer lines (Fig. 121 E. F., 
etc.). 




Fig. 12- 



Fig. 124. 



Fig. 125. 



Fig 123. ( 1). Section of the tube and stage of the microscope with the spectral 
ocular or micro-spectroscope in position. 

Amici Prism ($ iSS). — The direct vision prism of Amici in which the central 
shaded p> ism of flint glass gives the dispersion or separation into colors, while the 



CH. VI~\ MICRO-SPECTROSCOPE AND POLAR/SCOPE 141 

end prisms of crown glass cause the rays to emerge approximately parallel with 
the axis of the microscope. A single ray is represented as entering the prism and 
this is divided into three groups {Red, Yellow, Blue), which emerge from the 
prism, the red being least and the blue most bent toward the base of the flint prism 
(see Fig. 124). 

Hinge. — The hinge on which the prism tube turns when it is swung off the 
ocular. 

Ocular {\ 188) — The ocular in which the slit mechanism takes the place of the 
diaphragm (§ 198). The eye-lens is movable as in a micrometer ocular, so that 
the slit may be accurately focused for the different colors {\ 200). 

5. Screw for setting the scale of wave lengths { \ 202). 

S' ' . Screw for regidating the width of the slit ( \ 198). 

S". Screw for clamping the micro-spectroscope to the tube of the microscope. 

Scale Tube. — The tube near the tipper end containing the Angstrom scale and 
the lenses for projecting the image upon the upper face of the Amici prism , whence 
it is reflected upward to the eye with the different colored rays. At the right is a 
special mirror for lighting the scale. 

Slit. — The linear opening between the knife edges. Through the slit the light 
passes to the prism. It must be arra?iged parallel with the refracting edge of the 
prism, a?id of such a width that the Fraunhofer or Fixed Lines are very clearly and 
sharply defined when the eye-lens is properly Jocused ($ 198-200). 

Stage. — The stage of the microscope. This supports a watch-glass with sloping 
sides for containing the colored liquid to be examined. 

(3) Comparison Prism with tube for colored liquid (C L.), and mirror. The 
prism reflects horizontal rays vertically , so that when the prism is made to cover 
part of the slit two parallel spectra may be seen, one from light sent directly through 
the entire microscope and one from the light reflected upward from the comparison 
prism. 

(4) View of the Slit Mechanism from below. — Slit, the linear space between 
the knife edges through which the light passes. 

P. Comparison prism beneath the slit and covering part of it at will. 

S. S / . Screws for regulating the length and width of the slit. 

Fig. 124. Flint-Glass Prism showing the separation or dispersion of while 
light into the three groups of colored rays {Red, Yellow, Blue), the blue rays being 
bent the most from the refracting edge {\ 189). 

Fig. 125. Sectional View of a Microscope with the Polariscope in position 
(§218-227). 

Analyzer and Polarizer. — They are represented with corresponding faces par- 
allel so that the polarized beam could traverse freely the analyzer. If either Nicol 
were rotated 90 they would be crossed and no light would traverse the analyzer 
unless some polarizing substance were used as object, (a) Slot in the analyzer 
tube so that the analyzer may be raised or lowered to adjust it for difference of 
level of the eye-point in different oculars {\ 59, 200). 

Pointer and Scale. — The pointer attached to the analyzer and the scale or divided 
circle clamped {by the screw S) to the tube of the microscope. The pointer and 
scale enable one to determine the exact amount of rotation of the analyzer {\ 220). 

Object — The object whose character is to be investigated by polarized light. 



142 MICRO-SPECTROSCOPE AND POLARISCOPE [CH. VI 

To show the necessity of focusing the slit, move the eye-lens down 
or up as far as possible, and the Fraunhofer lines cannot be seen. 
While looking into the spectroscope move the ocular lens up or down, 
and when it is focused the Fraunhofer lines will reappear. As the dif- 
ferent colors of the spectrum have different wave lengths, it is neces- 
sary to focus the slit for each color if the sharpest possible pictures are 
desired. 

It will be found that the eye-lens of the ocular must be farther 
from the slit for the sharpest focus of the red end than for the sharpest 
focus of the lines at the blue end. This is because the wave length of 
red is markedly greater than for blue light. 

Longitudinal dark lines of the spectrum may be due to irregular- 
ity of the edge of the slit or to the presence of dust. They are most 
troublesome with a very narrow slit. 

§ 201. Comparison or Double Spectrum. — In order to com- 
pare the spectra of two different substances it is desirable to be able to 
examine their spectra side by side. This is provided for in the better 
forms of micro-spectroscopes by a prism just below the slit, so placed 
that the light entering it from a mirror at the side of the drum shall be 
totally reflected in a vertical direction, and thus parallel with the ra} T s 
from the microscope. The two spectra will be side by side with a 
narrow dark line separating them. If now the slit is well focused and 
da}dight be sent through the microscope and into the side to the reflect- 
ing or comparison prism, the colored bands and the Fraunhofer dark 
lines will appear directly continuous across the two spectra. The 
prism for the comparison spectrum is movable and may be thrown en- 
tirely out of the field if desired. When it is to be used, it is moved 
about half way across the field so that the two spectra shall have 
about the same width. 

§ 202. Scale of Wave Lengths. — In the Abbe micro-spectro- 
scope the scale is in a separate tube near the top of the prism and at 
right angles to the prism-tube. A special mirror serves to light the 
scale, which is projected upon the spectrum by a lens in the scale-tube. 
This scale is of the Angstrom form, and the wave lengths of any part 
of the spectrum may be read off directly, after the scale is once set in 
the proper position, that is, when it is set so that any given wave 
length on the scale is opposite the part of the spectrum known by pre- 
vious investigation to have that particular wave length. The point 
most often selected for setting the scale is opposite the sodium line 
where the wave length is, according to Angstrom, 0.5892 /x. In ad- 



CH. Vr\ MICRO-SPECTROSCOPE AND POLAR/SCOPE 143 

lusting the scale, one may focus very sharply the dark sodium line of 
the solar spectrum and set the scale so that the number 0.589 is oppo- 
site the sodium or D line, or a method that is frequently used and 
serves to illustrate § 191-2, is to sprinkle some salt of sodium (carbon- 
ate of sodium is good) in an alcohol lamp flame and to examine this 
flame. If this is done in a darkened place with a spectroscope, a 
narrow bright band will be seen in the yellow part of the spectrum. 
If now ordinary daylight is sent through the comparison prism, the 
bright line of the sodium will be seen to be directly continuous with 
the dark line at D in the solar spectrum (Fig. 121). By reflecting 
light into the scale-tube the image of the scale will appear on the 
spectrum, and by a screw just under the scale-tube but within the 
prism-tube, the proper point on the scale (0.589 pi) can be brought 
opposite the sodium band. All the scale will then give the wave 
lengths directly. Sometimes the scale is oblique to the spectrum. 
This ma} T be remedied by turning the prism-tube slightly one way or 
the other. It may be due to the wrong position of the scale itself. If 
so, grasp the milled ring at the distal end of the scale-tube and, while 
looking into the spectroscope, rotate the tube until the lines of the scale 
are parallel with the Fraunhofer lines. It is necessary in adjusting 
the scale to be sure that the larger number, 0.70, is at the red end of 
the spectrum. 

The numbers on the scale should be very clearly defined. If they 
do not so appear, the scale-tube must be focused by grasping the outer 
tube of the scale-tube and moving it toward or from the prism-tube 
until the scale is distinct. In focusing the scale, grasp the outer scale- 
tube with one hand and the prism-tube with the other, and push or 
pull in opposite directions. In this way one will be less liable to injure 
the spectroscope. 

§ 203. Designation of Wave Length. — Wave lengths of light 
are designated by the Greek letter X, followed b} r the number indicat- 
ing the wave length in some fraction of a meter. With the Abbe 
micro- spectroscope the micron is taken as the unit as with other micro- 
scopical measurements (§ 166). Various units are in use, as the one 
hundred thousandth of a millimeter, millionths or ten millionths of a 
millimeter. If these smaller units are taken, the wave lengths will be 
indicated either as a decimal fraction of a millimeter or as whole num- 
bers. Thus, according to Angstrom, the wave length of sodium light 
is 5892 ten millionths mm., or 589.2 millionths, or 58.92 one hundred 
thousandths, or 0.5892 one thousandth mm., or 0.5892 pi. The last 
would be indicated thus, AD= 0.5892 pi. 



144 MICRO-SPECTROSCOPE AND POLAR/SCOPE \CH. VI 

§ 204. Lighting for Micro-spectroscope. — For opaque objects 
a strong light should be thrown on them either with a concave mirror 
or a condensing lens. For transparent objects the amount of the sub- 
stance and the depth of color must be considered. As a general rule 
it is well to use plent3^ of light, as that from an Abbe illuminator with 
a large opening in the diaphragm, or with the diaphragm entirely 
open. For very small objects and thin. layers of liquids it may be 
better to use less light. One must try both methods in a given case, 
and learn by experience. 

The direct and the comparison spectra should be about equally 
illuminated. One can manage this by putting the object requiring the 
greater amount of illumination on the stage of the microscope and 
lighting it with the Abbe illuminator. In lighting it is found in gen- 
eral that for red or yellow objects, lamp- light gives very satisfactory 
results. For the examination of blood and blood crystals the light 
from a petroleum lamp is excellent. For objects with much blue or 
violet, daylight or artificial light rich in blue light is best. 

Furthermore, one should be on his guard against confusing the 
ordinary absorption bands with the Fraunhofer lines when daylight is 
used. With lamp-light the Fraunhofer lines are absent and, therefore, 
not a source of possible confusion. 

§ 205. Objectives to Use with the Micro-spectroscope. — If 
the material is of considerable bulk, a low objective (16 to 50 mm.) is 
to be preferred. This depends on the nature of the object under ex- 
amination, however. In case of individual crystals one should use 
sufficient magnification to make the real image of the crystal entirely 
fill the width of the slit. The length of the slit may then be regulated 
by the screw on the side of the drum, and also by the comparison 
prism. If the object does not fill the whole slit the white light enter- 
ing the spectroscope with the light from the object might obscure the 
absorption bands. For opaque objects illuminating objectives are 
useful (§28, 233.) 

In using high objectives with the micro-spectroscope one must 
very carefully regulate the light ( Ch. II) and sometimes shade the 
object. 

§ 206. Focusing the Objective. — For focusing the objective the 
prism-tube is swung aside, and then the slit made wide by turning the 
adjusting screw at the side. If the slit is open one can see objects 
when the microscope is focused as with an ordinary ocular. After an 
object is focused, it may be put exactl} r in position to fill the slit of the 



CH. Vr\ MICRO-SPECTROSCOPE AND POLAR/SCOPE 1 45 

spectroscope, then the knife edges are brought together till the slit is 
of the right width ; if the slit is then too long it may be shortened by 
using one of the mechanism screws on the side, or if that is not suffi- 
cient, by bringing the comparison prism farther over the field. If one 
now replaces the Amici prism and looks into the microscope, the 
spectrum is liable to have longitudinal shimmering lines. To get rid 
of these focus up or down a little so that the microscope will be 
slightly out of focus. 

§ 207. Amount of Material Necessary for Absorption Spectra 
and its Proper Manipulation. — The amount of material necessary to 
give an absorption spectrum varies greatly with different substances, 
and can be determined only by trial. Ii a transparent solid is under 
investigation it is well to have it in the form a wedge, then succes- 
sive thicknesses can be brought under the microscope. If a liquid sub- 
stance is being examined, a watch glass with sloping sides forms an 
excellent vessel to contain it, then successive thicknesses of the liquid 
can be brought into the field as with the wedge-shaped solid. Fre- 
quently only a very weak solution is obtainable ; in this case it can be 
placed in a homoeopathic vial, or in some glass tubing sealed at the 
end, then one can look lengthwise through the liquid and get the 
effect of a more concentrated solution. For minute bodies like crystals 
or blood corpuscles, one may proceed as described in the previous 
section. 

MICRO-SPECTROSCOPE — EXPERIMENTS* 

^ 208. Put the micro-spectroscope in position, arrange the slit 
and the Amici prism so that the spectrum will show the various spec- 
tral colors going directly across it (§ 198-199) and carefully focus the 
slit. This may be done either by swinging the prism-tube aside and 
proceeding as for the ocular micrometer (§ 172), or by moving the 
eye- lens of the ocular up and down while looking into the micro- 
spectroscope until the dark lines of the solar spectrum are distinct. If 
they cannot be made distinct by focusing the slit, then the light is too 
feeble or the slit is too wide (§ 198). With the lever move the com- 
parison prism across half the field so that the two spectra shall be of 
about equal width. For lighting, see § 204. 



*If one does not possess a micro-spectroscope, quite satisfactory results may be 
obtained by using a microscope with a 16 to 12 mm. objective and a pocket, direct- 
vision spectroscope in place of the eye-piece. (Bleile, Trans. Amer. Micr. Soc. 
1900, p. 8). 



146 MICRO-SPECTROSCOPE AND POLAR/SCOPE [CH. VI 

§ 209. Absorption Spectrum of Permanganate of Potash. — 
Make a solution of permanganate of potash in water of such a strength 
that a stratum 3 or 4 mm. thick is transparent. Put this solution in a 
watch-glass with sloping sides, and put it under the microscope. Use 
a 50 mm. or 16 mm. objective, and use the full opening of the illumi- 
nator. Light strongly. Look into the spectroscope and slowly move 
the watch-glass into the field. Note carefully the appearance with the 
thin stratum of liquid at the edge and then as it gradually thickens on 
moving the watch-glass still farther along. Count the absorption 
bands and note particularly the red and blue ends. Compare carefully 
with the comparison spectrum (Figs. 121, 122). For strength of solu- 
tion see § 207. 

§ 210. Absorption Spectrum of Blood. — Obtain blood from a 
recently killed animal, or flame a needle, and after it is cool prick the 
finger two or three times in a small area, then wind a handkerchief or 
a rubber tube around the base of the finger, and squeeze the finger 
with the other hand. Some blood will ooze out of the pricks. Rinse this 
off into a watch-glass partly filled with water. Continue to add the blood 
until the water is quite red. Place the watch-glass of diluted blood un- 
der the microscope in place of the permanganate, using the same object- 
ive, etc. Note carefully the spectrum. It would be advantageous to 
determine the wave length opposite the center of the dark bands. This 
may easily be done by setting the scale properly as described in § 202. 
Make another preparation, but use a homeopathic vial instead of a 
watch-glass. Cork the vial and lay it down upon the stage of the 
microscope. Observe the spectrum. It will be like that in the watch- 
glass. Remove the cork and look through the whole length of the vial. 
The bands will be much darker, and if the solution is thick enough 
onfy red and a little orange will appear. Re-insert the cork and incline 
the vial so that the light traverses a very thin layer, then gradually 
elevate the vial and the effect of a thicker and thicker layer may be 
seen. Note especially that the two characteristic bands unite and 
form one wide band as the stratum of liquid thickens. Compare with 
the following : 

Add to the vial of diluted blood a drop or two of ammonium sul- 
phide, such as is used for a reducing agent in chemical laboratories. 
Shake the bottle gentl} 7 and then allow it to stand for ten or fifteen 
minutes. Examine it and the two bands will have been replaced by a 
single, less clearly defined band in about the same position. The 
blood will also appear somewhat purple. Shake the vial vigorously 



CH. VI] MICRO-SPECTROSCOPE AND POLARISCOPE 147 

and the color will change to the bright red of fresh blood. Examine 
it again with the spectroscope and the two bands will be visible. After 
five or ten minutes another examination will show but a single band. 
Incline the bottle so that a thin stratum may be examined. Note 
that the stratum of liquid must be considerably thicker to show the 
absorption band than was necessary to show the two bands in the first 
experiment. Furthermore, while the single band may be made quite 
black on thickening the stratum, it will not separate into two bands 
with a thinner stratum. In this experiment it is very instructive to 
have a second vial of fresh diluted blood, say that from the watch- 
glass, before the opening of the comparison prism. The two banded 
spectrum will then be in position to be compared with the spectrum of 
the blood treated with the ammonium sulphide. 

The two banded spectrum is that oi oxy-hemoglobin, or arterial blood, 
the single banded spectrum of hemoglobin (sometimes called reduced 
hemoglobin) or venous blood, that is, the respiratory oxygen is present 
in the two banded spectrum but absent from the single banded spectrum. 
When the bottle was shaken the hemoglobin took up oxygen from the 
air and became oxy-hemoglobin, as occurs in the lungs, but soon the 
ammonium sulphide took away the respiratory oxygen, thus reducing 
the oxy-hemoglobin to hemoglobin. This may be repeated many 
times (Fig. 122). 

§ 211. Met-Hemoglobin. — The absorption spectrum of met- 
hemoglobin is characterized by a considerable darkening of the blue 
end of the spectrum and of four absorption bands, one in the red near 
the line C and two between D and E, nearly in the place of the two 
bands of oxy-hemoglobin ; finally there is a somewhat faint, wide 
band near F. Such a met-hemoglobin spectrum is best obtained by 
making a solution of blood in water of such a concentration that the 
two oxy-hemoglobin bands run together (§ 210), and then adding 
three or four drops of a jo P er cent, aqueous solution of permanganate 
of potash. Soon the bright red will change to a brownish color, when 
it may be examined (Fig. 121). Instead of the permanganate one 
may use hydrogen dioxide (H 2 2 ). 

§ 212. Carbon Monoxide Hemoglobin (CO-Hemoglobin). — 
To obtain this, kill an animal in illuminating gas, or one may allow 
illuminating gas to bubble through some blood already taken from the 
body. The gas should bubble through a minute or two. The oxygen 
will be displaced by carbon monoxide. This forms quite a stable com- 
pound with hemoglobin, and is of a bright cherry-red color. Its 



148 MICRO-SPECTROSCOPE AND POLAR/SCOPE {CH. VI 

spectrum is nearly like that of oxy-hemoglobin, but the bands are 
farther toward the blue. Add several drops of ammonium sulphide and 
allow the blood to stand some time. No reduction will take place, 
thus forming a marked contrast to solutions of oxy-hemoglobin. By 
the addition of a few drops of glacial acetic acid a dark brownish red 
color is produced. 

§ 213. Carmine Solution. — Make a solution of carmine by put- 
ting -fVth gram of carmine in 100 cc. of water and adding 10 drops of 
strong ammonia. Put some of this in a watch-glass or in a small vial 
and compare the spectrum with that of oxy-hemoglobin or carbon 
monoxide hemoglobin. It has two bands in nearly the same position, 
thus giving the spectrum a striking similarity to blood. If now several 
drops, 15 or 20, of glacial acetic acid are added to the carmine, the 
bands remain and the color is not markedly changed, while with 
either oxy-hemoglobin or CO-hemoglobin the color would be de- 
cidedly changed from the bright red to a dull reddish brown, and 
the spectrum, if any could be seen, would be markedly different. 
Carmine and O-hemoglobin can be distinguished by the use of ammo- 
nium sulphide, the carmine remaining practically unchanged while the 
blood shows the single band of hemoglobin (§ 210). The acetic acid 
serves to differentiate the CO-hemoglobin as well as the O-hemoglobin. 

§ 214. Colored Bodies not giving Distinctly Banded Absorp- 
tion Spectra. — Some quite brilliantly colored objects, like the skin of 
a red apple, do not give a banded spectrum. Take the skin of a red 
apple, mount it on a slide, put on a cover-glass and add a drop of 
water at the edge of the cover. Put the preparation under the micro- 
scope and observe the spectrum. Although no bands will appear, in 
some cases at least, yet the ends of the spectrum will be restricted and 
various regions of the spectrum will not be so bright as the comparison 
spectrum. Here the. red color arises from the mixture of the unab- 
sorbed waves, as occurs with other colored objects. In this case, 
however, not all the light of a given wave length is absorbed, conse- 
quently there are no clearly defined dark bands, the light is simply 
less brilliant in certain regions and the red rays so predominate that 
they give the prevailing color. 

§ 215. Nearly Colorless Bodies with Clearly Marked Ab- 
sorption Spectra. — In contradistinction to the brightly colored 
objects with no distinct absorption bands are those nearly colorless 
bodies and solutions which give as sharply defined absorption bands as 
could be desired. The best examples of this are afforded b3^ solutions 



CH. VI] MICRO-SPECTROSCOPE AND POLARISCOPE 149 

of the rare earths, didymium, etc. These in solutions that give 
hardly a trace of color to the eye give absorption bands that almost 
rival the Fraunhofer lines in sharpness. 

§216. Absorption Spectra of Minerals. — As example take some 
monazite sand on a slide and either mount it in balsam (see § 256), 
or cover and add a drop of water. The examination maj^ be made also 
with the dry sand, but it is less satisfactory. Light well with trans- 
mitted light, and move the preparation slowly around. Absorption 
bands will appear occasionally . Swing the prism tube off the ocular, 
open the slit and focus the sand. Get the image of one or more grains 
directly in the slit, then narrow and shorten the slit so that no light 
can reach the spectroscope that has not traversed the grain of sand. 
The spectrum will be satisfactory under such conditions. It is 
frequently of great service in determining the character of unknown 
mineral sands to compare the spectra with known minerals. If the 
absorption bands are identical, it is strong evidence in favor of the 
identity of the minerals. For proper lighting see § 204. 

§ 217. While the study of absorption spectra gives one a great 
deal of accurate information, great caution must be exercised in draw- 
ing conclusions as to the identity or even the close relationship of 
bodies giving approximately the same absorption spectra. The rule 
followed by the best workers is to have a known body as control and 
to treat the unknown body and known body with the same reagents, 
and to dissolve them in the same medium. If all the reactions are 
identical then the presumption is strong that the bodies are ident- 
ical or very closely related. For example, while one might be in doubt 
between a solution of oxy- or CO-hemoglobin and carmine, the addition 
of ammonium sulphide serves to change the double to a single band 
in the O-hemoglobin, and glacial acetic acid enables one to distinguish 
between the CO-blood and the carmine, although the ammonium sul- 
phide would not enable one to make the distinction. Furthermore it 
is unsafe to compare objects dissolved in different media. The same 
objects as "cyanine and aniline blue dissolved in alcohol give a very 
similar spectrum, but in water a totally different one." "Totally dif- 
ferent bodies show absorption bands in exactly the same position (solid 
nitrate of uranium and permanganate of potash in the blue)." (Mac- 
Munnj. The rule given by MacMunn is a good one : "The recogni- 
tion of a body becomes more certain if its spectrum consists of several 
absorption bands, but even the coincidence of these bands with those 
of another body is not sufficient to enable us to infer chemical identity; 



150 MICRO-SPECTROSCOPE AND POLARISCOPE [CH. VI 

what enables us to do so with certainty is the fact : that the two solu- 
tions give bands of equal inte?isities in the same parts of the spectrum 
which undergo analogous changes on the addition of the same reagent. ' ' 

REFERENCES TO THE MICRO-SPECTROSCOPE AND 
SPECTRUM ANALYSIS 

The micro-spectroscope is playing an ever-increasingly important role in the 
spectrum analysis of animal and vegetable pigments, and of colored mineral and 
chemical substances, therefore a somewhat extended reference to literature will be 
given. Full titles of the books and periodicals will be found in the Bibliography 
at the end. 

Angstrom, Recherches sur le spectre solaire, etc. Also various papers in 
periodicals. See Royal Soc's Cat'l Scientific Papers ; Anthony & Brackett ; Beale, 
p. 269 ; Behrens, p. 139 ; Kossel und Schiefferdecker, p. 63 ; Carpenter, p. 323 ; 
Browning, How to Work with the Spectroscope, and in Monthly Micr. Jour., II, 
p. 65 ; Daniell, Principles of Physics. The general principles of spectrum analysis 
are especially well stated in this work, pp. 435-455 ; Davis, p. 342 ; Dippel, p. 
277 ; Frey ; Gamgee, p. 91 ; Halliburton ; Hogg, p. 122 ; also in Monthly Micr. 
Jour., Vol. II, on colors of flowers; Jour, Roy. Micr. Soc, 1880, 1883, and in 
various other vols.; Kraus ; L,ockyer ; M'Kendrick ; MacMunn ; and also in 
Philos, Trans. R. S. , 1S86 ; various vols, of Jour. Physiol.; Nageli und Schwend- 
ener ; Proctor ; Ref. Hand-Book Med. Science, Vol. I, p. 577, VI, p. 516, VII, p. 
426; Roscoe ; Schellen ; Sorby, in Beale, p. 269; also Proc. R. S., 1874, p. 31, 
1867, p. 433 ; see also in the Scientific Review, Vol. V, p. 66, Vol. II, p. 419. 
The larger works on Physiology, Chemistry and Physics may also be consulted 
with profit. 

Vogel, Spectrum analysis ; also in Nature, Vol. xix, p. 495, on absorption spec- 
tra. The bibliography in MacMunn is excellent and extended. 

For hemochromogen in medico-legal cases see Bleile, Trans. Amer. Micr. 
Soc, 1900, p. 9. 

MICRO-POLARISCOPE 

\ 21S. The micro-polariscope, or polarizer, is apolariscope used in connection 
with a microscope. 

The most common and typical form consists of two Nicol prisms, that is, two 
somewhat elongated rhombs of Iceland spar cut diagonally and cemented together 
with Canada balsam. These Nicol prisms are then mounted in such a way that 
the light passes through them lengthwise, and in passing is divided into two rays 
of plane polarized light. The one of these rays obeying most nearly the ordinary 
law of refraction is called the ordinary ray, the one departing farthest from the 
law is called the extra-ordinary ray. These two rays are not only polarized, but 
polarized in planes almost exactly at right angles to each other. The Nicol prism 
totally reflects the ordinary ray at the cemented surface as it meets that surface at 
an angle greater than the critical angle, and only the extraordinary or less refracted 
ray is transmitted. 



CH. VI} MICRO-SPECTROSCOPE AND POLARISCOPE 151 

\ 219. Polarizer and Analyzer. — The polarizer is one of the Nicol prisms. It is 
placed beneath the object and in this way the object is illuminated with polarized 
light. The analyzer is the other Nicol and is placed at some level above the object, 
very conveniently above the ocular. 

When the corresponding faces of the polarizer and analyzer are parallel i. e., 
when the faces through which the oblique section passes are parallel, light passes 
freely through the analyzer to the eye. If these corresponding faces are at right 
angles, that is, if the Nicols are crossed, then the light is entirely cut off and the 
two transparent prisms become opaque to ordinary light. There are then, in the 
complete revolution of the analyzer, two points, at o° and 180 , where the corre- 
sponding faces are parallel and where light freely traverses the analyzer. There 
are also two crossing points of the Nicols, at 90 and 270 , where the light is extin- 
guished. In the intermediate points there is a sort of twilight. 

\ 220. Putting the Polarizer and Analyzer in Position. — Swing the diaphragm 
carrier of the Abbe illuminator out from under the illuminator, remove the disk 
diaphragm or open widely the iris diaphragm and place the analyzer in the dia- 
phragm carrier, then swing it back under the illuminator. Remove the ocular, 
put the graduated ring on the top of the tube and then replace the ocular and put 
the analyzer over the ocular and ring. Arrange the graduated ring so that the indi- 
cator shall stand at o° when the field is lightest. This may be done by turning the 
tube down so that the objective is near the illuminator, then shading the stage so 
that none but polarized light shall enter the microscope. Rotate the analyzer until 
the lightest possible point is found, then rotate the graduated ring till the index 
stands at 0°. The ring may then be clamped to the tube by the side screw for the 
purpose. Or, more easily, one may set the index at o°, clamp the ring to the 
microscope, then rotate the draw-tube of the microscope till the field is lightest. 

\ 221. Adjustment of the Analyzer. — The analyzer should be capable of 
moving up and down in its mounting, so that it can be adjusted to the eye-point 
of the ocular with which it is used. If on looking into the analyzer with parallel 
Nicols the edge of the field is not sharp, or if it is colored, the analyzer is not in 
a proper position with reference to the eye-point, and should be raised or lowered 
till the edge of the field is perfectly sharp and as free from color as the ocular 
with the analyzer removed. 

\ 222. Objectives to Use with the Polariscope. — Objectives of the lowest pow- 
ers may be used, and also all intermediate forms up to a 2 mm. homogeneous im- 
mersion. Still higher objectives may be used if desired. In general, however, 
the lower powers are somewhat more satisfactory. A good rule to follow in this 
case is the general rule in all microscopic work, — use the power that most clearly 
and satisfactorily shows the object under investigation. 

\ 223. Lighting for Micro-Polariscope Work. — Follow the general directions 
given in Chapter II. It is especially necessary to shade the object so that no un- 
polarized light can enter the objective, otherwise the field cannot be sufficiently 
darkened. No diaphragm is used over the polarizer for most examinations. Direct 
sunlight may be used to advantage with some objects, and as a rule the object 
would best be very transparent. 

\ 224. Mounting Objects for the Polariscope. — So far as possible objects 
should be mounted in balsam to render them transparent. In many cases objects 
mounted in water do not give satisfactory polariscope appearances. For example, 



152 MICRO-SPECTROSCOPE AND POLAR/SCOPE \CH. VI 

if starch is mounted dry or in water, the appearances are not so striking as in a 
balsam mount (Davis, p. 337 ; Suffolk). 

\ 225. Purpose of a Micro-Polariscope. — The object of a micro-polariscope is 
to determine, in microscopic masses, one or more of the following points : (A) 
Whether the body is singly refractive, mono-refringent, or isotropic, that is, opti- 
cally homogeneous, as are glass and crystals belonging to the cubical system ; (B) 
Whether the object is doubly refractive or anisotropic, uniaxial or biaxial ; (C) 
Pleochroism ; (D) The rotation of the plane of polarization, as with solutions of 
sugar, etc. ; (E) To aid in petrology and mineralogy ; (F) To aid in the determi- 
nation of very minute quantities of crystallizable substances; (G) For the pro- 
duction of colors. 

For petrological and mineralogical investigations the microscope should possess 
a graduated, rotating stage so that the object can be rotated, and the exact angle 
of rotation determined. Fig. 126. It is also found of advantage in investigating 
object swith polarized light where colors appear, to combine a polariscopic and 
spectroscope ( Spectro-Polariscope ) . 

MICRO-POLARISCOPE — EXPERIMENTS 

§ 226. Arrange the polarizer and analyzer as directed above (§ 220) 
and use a 16 mm. objective except when otherwise directed. 

(A) Isotropic or Singly Refracting Objects. — Light the mi- 
croscope well and cross the Nicols, shade the stage and make the field 
as dark as possible (§219). As an isotropic substance, put an ordin- 
ary glass slide under the microscope. The field will remain dark. As 
an example of a crystal belonging to the cubical system and hence iso- 
tropic, make a strong solution of common salt (sodium chloride) 
put a drop on a slide and allow it to crystallize, put it under the 
microscope, remove the analyzer, focus the crystals and then replace 
the analyzer and cross the Nicols. The field and the crystals will re- 
main dark. 

(B) Anisotropic or Doubly Refracting Objects. — Make a fresh 
preparation of carbonate of lime crystals like that described for pedesis 
(§ 151), or use a preparation in which the crystals have dried to the 
slide, use a 5 or 3 mm. objective, shade the object well, remove the 
analyzer and focus the crystals, then replace the anal} T zer. Cross the 
Nicols. In the dark field will be seen multitudes of shining crystals, 
and if the preparation is a fresh one in water, part of the smaller 
crystals will alternately flash and disappear. By observing carefully, 
some of the larger crystals will be found to remain dark with crossed 
Nicols, others will shine continuously. If the crystals are in such a 
position that the light passes through them parallel with the optic 



Fig. 126. ChamoVs Microscope 
for Micro-Chemical Analysis {Jour- 
nal of Applied Microscopy \ 1899, p. 

503'. 

This is a modified and simplified 

pelrographical microscope and has 
all the attachments and motions nec- 
essary for micro-chemical analysis. 
As the objects studied are mostly 
liquid or in liquids the microscope 
has no joint as it must be used in a 
vertical position. 




154 MICRO-SPECTROSCOPE AND POLAR/SCOPE \_CH. VI 

axis,* the crystals are isotropic like salt crystals and remain dark. 
If, however, the light traverses them in any other direction the ray 
from the polarizer is divided into two constituents vibrating in planes 
at right angles to each other, and one of these will traverse the an- 
alyzer, hence such crystals will appear as if self-luminous in a dark 
field. The experiment with these crystals from the frog succeeds well 
with a 2 mm. homogeneous immersion. 

As a further illustration of anisotropic objects, mount some cotton 
fibers in balsam (§ 256), also some of the lens paper (§ 114;. These 
furnish excellent examples of vegetable fibers. 

Striated muscle fibers are also very well adapted for polarizing 
objects. 

As examples of biaxial crystals, allow some borax solution to dry 
and oystallize on a slide ; use the crystals as objects. As all doubly 
refracting objects restore the light with crossed Nicols, they are some- 
times called depolarizing. 

(C) Pleochroism. — This is the exhibition of different tints as the 
analyzer is rotated. An excellent subject for this will be found in 
blood crystals. 

§ 227. Production of Colors. — For the production of gorgeous 
colors, a plate of selenite giving blue and yellow colors is placed between 
the polarizer and the object. If properly mounted, the selenite is very 
conveniently placed on the diaphragm carrier of the Abbe illuminator, 
just above the polarizer. A thin plate or film of mica also answers 
well. 

It is not necessary to use selenite or mica for the production of the 
most glorious colors in many objects. One of the most beautiful pre- 
parations, and one of the most instructive also, may be prepared as 
follows : Heat some xylene balsam on a slide until the xylene is nearly 
evaporated. Add some crystals of the medicine, sulphonal and warm 
till the sulphonal is melted add mixes with the balsam. While the 
balsam is still melted put on a cover-glass. If one gets perfect crystals 
there will be shown not only the most beautiful colors, but the black 
cross with perfection. (Clark). 



*The optic axis of doubly refracting crystals is the axis along which the crystal 
is not doubly refracting, but isotropic like glass. When there is but one such 
axis, the crystal is said to be uniaxial, if there are two such axes the crystal is 
said to be bi-axial. 

The crystals of carbonate of lime from the frog (see § 151 ) are uniaxial crystals. 
Borax crystals are bi-axial. 



CH. VI} MICRO-CHEMISTRY 155 

It is very instructive and interesting to examine many organic and 
inorganic substances with a micro-polarizer. 

REFERENCES TO THE POLARISCGPE AND TO THE USE OF POLARIZED 

LIGHT. 

Anthony & Brackett, 133 ; Behrens ; Behrens, Kossel und Schiefferdecker ; Car- 
noy, 61 ; Carpenter-Dallinger, 317, 1097 ; Clark ; Daniell, 494 ; Davis ; v. Ebener ; 
Gamgee ; Halliburton, 36, 272 ; Hogg, 133, 729 ;.Lehmann ; M'Kendrick ; Nageli 
und Schwendener, 299; Quekett ; Suffolk, 125 ; Valentin. Physical Review, I., p. 
127. Daniell, Physics for Medical Students. Nichols, Physics. 

MICRO-CHEMISTRY 

§ 228. During the last decade the microscope has become one of 
the necessities of the expert chemist, and the signs of the times indicate 
that in every research laboratory of chemistry the microscope will be- 
come as familiar as it now is in research laboratories of biology. Its 
proper place in chemistry has been admirably stated by Chamot : 

" It is rather remarkable how slow American chemists have been in realizing 
the importance of the microscope as an adjunct to every chemical laboratory. 
This is, perhaps, largely due to the fact that few of our students in chemistry 
become familiar with the construction and manipulation of this instrument, just 
as few of them become sufficiently familiar with the spectroscope and its manifold 
uses ; and doubtless also because of the prevailing impression that a microscope is 
primarily an instrument for the biologist and is of necessity a most expensive 
luxury. The fact is, however, that this instrument is now far from being a luxury 
to the chemist, and the time is not far distant when it will be conceded to be as 
much a necessity in every analytical laboratory as is the balance. 

" Nor is the apprenticeship to its use in chemical work long nor intricate. 

" Micro-chemical analysis should appeal to every chemist because of its neat- 
ness, wonderful delicacy, in which it is not excelled even by the spectroscope, and 
the expedition with which an analysis can be made. A complete analysis, intricate 
though it may be, is a matter of a few minutes rather than of a few hours. 

" While there is no good reason to believe, as do some enthusiasts, that this 
new system is to displace the old analysis in the wet way, every chemist should, 
nevertheless, familiarize himself with the microscope, its accessories, and the 
elegant and time-saving methods of micro-analysis, thus enabling him to examine 
qualitatively the most minute amounts of material with a rapidity and accuracy 
which is truly marvelous ; not to speak of the many substances for which no other 
method of identification is known. ♦ 

• " At present the greatest bar to its general use is the absence of any well 
defined scheme,. and the absolute necessity of being well grounded in general 
chemistry. There are no tables which can be followed in a mechanical way by 
the student, but on the contrary he is obliged to exercise his knowledge and judg- 
ment at every step. For this very reason the introduction of this subject into the 
list of those now taught is greatly to be desired." 



156 MICRO-CHEMISTRY [CH. VI 

It will be seen by the last paragraph that in chemistry as in biology, 
the microscope is only an aid to investigation ; it cannot take the place of 
thorough training in the fundamentals of the subject under investiga- 
tion. 

§ 229. The following list of substances is suggested by Dr. 
Chamot for beginning practice as the results given are definite and 
easilv obtained : 



SUGGESTIONS FOR STUDY ON CRYSTAL SYSTEMS 



'Isometric. 



Sodium chlorid, potassium chlorid, potassium iodid. 

Alums, crystallize in octahedra, cubes or combinations of the two. It is 
well to recall that the alums have the general formula, M 2 (S0 4 ) 3 . N 2 S0 4 24 
H 2 0, where -M- can be Al, Cr, Mn, Fe, In, Ga, Tl, and -N- Na, K, Rb, 
Cs, NH, Ag, or Tl. All alums are isomorphous. 

Tetragonal. 

Potassium copper chlorid. Ammonium copper chlorid. 
Nickel sulfate 6H 2 0. This salt is dimorphic, crystallizing also in the mono- 
clinic system. Nickel sulfate 7H 2 is orthorhombic. 

Orthorhombic. 

Mercuric chlorid. Silver nitrate. Potassium sulfate. Potassium nitrate. 
Magnesium sulfate 7H 2 0. Potassium chromate. Sodium nitrate (also 
Hexagonal). 

Monoclinic. 

Potassium chlorate (sodium chlorate is Isomet. or Tetrag. ) 

Lead acetate. Copper acetate H 2 0. Oxalic acid. 

Ferrous sulfate, this salt forms normally with 7 H 2 and is then Mono- 
clinic, but in presence of zinc sulfate becomes Orthorhombic, and in presence 
of copper sulfate, Triclinic. 

Sodium sulfate ioH 2 0. 

Triclinic. 

Copper sulfate 5H 2 0. Boric acid. 
Hexagonal. 

Lead iodid. Sodium nitrate (also Orthorhombic). 

\ 230. Before performing analytical tests, it is necessary that the student 
be familiar with the properties of crystals and also thoroughly familiar with 
the appearance of crystals of the chlorides, nitrates, and sulfates of Sodium, 
Potassium, and Ammonium ; since some of these salts are sure to appear in 
almost every test drop examined. For this reason the following experiments 
should be carefully performed." 



CH. VI~\ MICRO-CHEMISTRY 157 

AN EXERCISE FOR PRACTICE 
1. "Sodium chlorid. Isometric system 

a. Take a fragment of the salt the size of this period (.) or a little 
smaller. Dissolve at the corner of a slide in a minute drop of distilled water. 
Heat over the "micro" flame so as to cause rapid evaporation. When dry 
examine residue (after cooling) with yi inch obj. and 2 in. eyepiece. 

Breathe on the preparation several times, allow the moisture to evaporate 
spontaneously. There should now be obtained well developed crystals instead 
of a mere crystalline mass. Too rapid crystallization is always to be avoided. 
The object of the experiment is to emphasize this fact. 

Select one of the best crystals. Measure its angles. Try its behavior 
between crossed Nicols. (§ 219. ) 

b. Place a small drop of water in the usual position add carefully Ferric 
•Chlorid till the drop when held over white paper appears distinctly 
yellow. Stir. Add two or three tiny fragments of Sodium Acetate at 
the center of the drop. Place on the stage of the microscope. There is 
formed Ferric acetate, Sodium chlorid and possibly a double chlorid of iron 
and sodium. Notice the following points. 1. Tendency toward formation 
of double salt. 2. That the type crystal of NaCl is a cube. 3, Cubes may 
so grow as to present the appearance of a rectangular prism. 4. In certain 
positions cubes have the appearance of a pyramid. 5. In other positions they 
exhibit a hexagonal outline, thus simulating a polyhedron of many faces. 
6. There is scarcely any tendency in this case toward the formation of the 
hoppers observed in the first experiment (a). 7. The crystals often develop 
fastest along the diagonal planes so that the regular faces are replaced by 
pyramidal depressions." 



Fig. 127. CzapskVs Ocular Iris-diaphragm with cross 
hairs for examining and accurately determining the axial im- 
ages of small crystals. The iris diaphragm enables the observer 
to make the field as large or small as desired. 

A. Lo7igitudinal section. 

B. Transection, showing the cross lines and the iris dia- 
phragm with the projecting part at the left, by which the dia- 
phragm is opened and closed. (Zeiss' Catalog). 



For directions and hints in micro-chemical work and crystallography, consult 
the various volumes of the Journal of the Roy. Micr. Soc. ; Zeitschrift fur physio- 
logische Chemie, and other chemical journals ; Wormly ; Klement & Renard ; 
Carpenter-Dallinger ; Hogg ; Behrens, Kossel und Schiefferdecker ; Frey ; Dana, 
and other works on mineralogy ; Davis. Behrens, T. H. — Anleitung zur micro- 
chemischen Analyse der wichtigsten organischen Verbindungen. Hamburg, 
1895-1897. Microchemische Technik, 2d edition, Hamburg, 1900. A manual of 
microchemical analysis with an introdnctory chapter by J. W. Judd, London, 




158 TEXTILE FIBERS [CH. VI 

1894. Especial attention is also called to the articles by Dr. E. M. Chamot in the 
Journal of Applied Microscopy beginning with vol. ii. p. 502, and continued in 
vol. iii and iv. 

TEXTILE FIBERS, FOOD AND PHARMACOLOGICAL PRODUCTS 

§ 231. The microscope is coming more and more into use for the 
determination of the character of textile fibers, both in the raw state 
and after manufacture. As the textile fibers have distinctive char- 
acters it is not difficult to determine mixtures in fabrics of various 
kinds. The student is advised to study carefully known fibers, as of 
cotton, wool, linen, silk, jute, etc., so that he is certain of the appear- 
ances, and then to determine of what fibers different fabrics are com- 
posed. He will be astonished at the amount of "Alabama wool" in 
supposedly all wool goods. 

For works and articles upon textile fibers see : Herzfeld, J. Trans- 
lated by Salter. The technical testing of yarns and textile fabrics 
with reference to official specifications. London, 1898. E. A. 
Posselt — The structure of fibers, yarns and fabrics. Philadelphia and 
and Dondon, 1891. Dr. C. Rougher — Des filaments vegetaux em- 
ployes dans l'industrie. Paris, 1873. Wm. P. Wilson and E. Fah- 
ring — ,The conditioning of wool and other fabrics in the technological 
laboratories of the Philadelphia Commercial Museum. Journal of Ap- 
plied Microscopy, Vol. II, (1899) pp. 290-292, 457-460. Bulletin of 
the National Association of Wool Growers, 1875, p. 470. Proceed- 
ings of the Amer. Micr. Soc, 1884, pp. 65-68. Besides these referen- 
ces one is liable to find pictures and discussions of various fibers in 
general works on the microscope, and in technical and general cyclo- 
paedias. 

§ 232. From the nature of food and pharmacological products 
adulterations are in many cases most accurately and easily determined 
by microscopic examination. The student will find constant reference 
to the microscopical characters of the genuine and spurious substances 
in medicines and other pharmacological products in works on pharmacy 
or pharmacology ; also in pharmacological journals and in druggists 
reports, e. g. the excellent and well illustrated article by Burt E. 
Nelson of the chemical laboratory of the Binghamton State Hospital 
on the Microscopical examination of tea, coffee, spices and condiments. 
This appeared in Merck's Report, Oct. 15, Dec. 15, 1898. The micro- 
scopical Journals also contain occasional articles bearing upon this 
subject. See also Food Products in bulletins of theU. S. Dep't Agr. 
Mace, E. — Des substances alimentaire, etc., Paris, 1891. Schimper, 



CH. VI] MICRO-METALLOGRAPHY 159 

A. F. W. Anleitung, etc. Jena, 1900. Hugh Gait, — The Microscopy 
of the starches, illustrated by photo-micrographs, London, 1900. 

THE MICROSCOPE IN METALLOGRAPHY 

§ 233. In the modern investigation of metals and alloys much 
light has been thrown upon the structural peculiarities which render 
some mixtures satisfactory and others unsatisfactory. There are two 
great methods : First, that of studying fractured surfaces without re- 
course to any reagents. Second, to polish a metallic surface carefully 
with emery or carborundum and finally with rouge or diamantine and 
then etch it with some acid for a longer or shorter time. For either 
method reflected light must be used. For low powers that obtained at a 
good window or by a lamp or a lamp and bulls eye are good. The illum- 
inating objectives (§ 28), i. e. objectives in which a prism in the side" 
of the objective reflects light down through the lenses which act as a 
condenser, are preferable for most work and indeed necessary if one uses 
high powers. For special microscope see Fig. 126 A. 

Elaborate arrangements have been devised for holding the piece 
of metal on the stage, but some beeswax, or some clay made plastic 
with glycerin answers well. For pictures of the appearances seen 
in studying metallic surfaces, see the journals of engineering and 
metallurgy, especially the Metallographist, a quarterly publication 
devoted to the study of metals with special reference to their physics 
and micro-structure, etc. In twenty-five or more of the great metal 
manufacturing establishments special laboratories for microscopic 
examination and investigation have been established. This is an illus- 
tration of what has frequently occurred — great manufacturing interests 
have outrun the universities in the appreciation and application of 
methods of reasearch. Fortunately, however, laboratories are already 
springing up in connection with the universities, and probably within 
ten j^ears every great technical school will have its laboratory of 
micro-metallography where students will have opportunity to perfect 
themselves in the preparation, photography and microscopic study of 
the metals and alloys. 

Beside the sources of information given above, see Dr. H. Ost und 
Dr. Fr. Kolbeck, Lehrbuch der chemischen Technologie mit einem 
Schlussabschnitt " Metallurgie." Hannover, 1901. Behrens, T. H. — 
Das mikroskopische Gefiige der Matalle, etc. Hamburg, 1894. For 
an excellent bibliography of 188 titles ; see the Metallographist, Vol. 
I, 1898, and appended to the special papers in all the volumes. Also 
in Iron Age, Jan. 27, 1898. Carpenter-Dallinger, p. 264. 



i6o 



MICR O-J/E TALLOGRAPHY 



[CH. VI 




Fig. 126 A. Microscope especially constructed for the study of metals and 
alloys. ( The Boston Testing Laboratories). 

The stage is rotary, and may be raised or lowered by rack and pinion. Above 
the objective is the arrangement for illumination {see Ch. VII). 



CHAPTER VII 



SLIDES AND COVER-GLASSES ; MOUNTING ; ISOLATION ; 
SECTIONING BY THE COLLODION AND THE PARAF- 
FIN METHODS ; LABELING AND STORING MICRO- 
SCOPICAL PREPARATIONS ; REAGENTS 



SLIDES AND COVER-GLASSES 

§ 234. Slides, Glass Slides or Slips, Microscopic Slides or Slips. — These are 
strips of clear flat glass upon which microscopic specimens are usually mounted for 
preservation and ready examination. The size that has been almost universally 
adopted for ordinary preparations is 25 x 76 millimeters (1x3 inches). For rock 
sections, slides 25 x 45 mm. or 32 x 32 mm. are used ; for serial sections, slides 
25 x 76 mm., 50 x 76 mm. or 37 x 87 mm. are used. For special purposes, slides 
of the necessary size are employed without regard to any conventional standard. 

Whatever size of slide is used, it should be made of clear glass and the edges 
should be ground. It is altogether false economy to mount microscopic objects 
on slides with unground edges. It is unsafe also as the unground edges are liable 
to wound the hands. 

For micro-chemical work Dr. Chamot recommends slides of half the length of 
those used in ordinary microscopic work. From the rapidity with which they are 
destroyed, he thinks the ground edges are unnecessarily expensive. He adds 
further: "It is a great misfortune that the colorless glass slips used in America 
and so excellent for ordinary microscopic work should be easily attacked by all 
liquids ; even water extracts a relatively enormous amount of alkalies and alka- 
line earths. The slips of greenish glass, while not as neat or desirable for general 
microscopy, seem to be decidedly more resistant, and are therefore preferable. " 
Transparent celluloid slides are recommended by Behrens for work where hydro- 
fluoric acid and its derivatives are to be examined. (Chamot, Jour. Appl. Micr. 
vol. iii, p. 793). 

\ 235. Cleaning Slides. — For new slides a thorough rinsing in clean water with 
subsequent wiping with a soft cloth like glass toweling, or thin cotton cloth like 
bleached cheese cloth (bunting or gauze, or absorbent surgical gauze), usually fits 
them for ordinary use. If they are not satisfactorily cleaned in this way, soak 
them a short time in 50% or 75% alcohol, let them drain for a few moments on a 
clean towel or on blotting paper, and then wipe with a soft cloth. In handling 
the slides grasp them by their edges to avoid soiling the face of the slide. After 
the slides are cleaned they should be stored in a place as free as possible from 
dust. For storing, covered glass dishes are best. Use museum jars or glass boxes 
(Fig. 150). 



l62 



SLIDES AND COVER-GLASSES 



\_CH. VII 



Fig. 128. Glass slide or slip of the ordinary size for microscopic work {3x1 in . 
j6 x 25 mm.). ( Cut loaned by the Spencer Lens Company). 



For old slides, if only water, glycerin or glycerin jelly has been used on them, 
they may be cleaned with water, or preferably, warm water and then with alcohol 
if necessary. Where balsam, or any oily or gummy substance has been used upon 
the slides, they may be freed from the balsam, etc., by soaking them for a week 
or more in one of the cleaning mixtures for glass. If they are first soaked in 
xylene, benzin or turpentine to dissolve the balsam, then soaked in the cleaning 
mixture, the time required will be much shortened (§ 242). After all foreign 
matter is removed the slides should be thoroughly rinsed in water to remove all 
the cleaning mixture. They may then be treated as directed for new slides. 

If slides with large covers, as in mounted series, are put into the cleaning 
mixture, the swelling of the balsam is liable to break the covers. Dissolving 
away the balsam with turpentine, avoids this, and greatly shortens the time neces- 
sary for cleaning the old slides and covers. 

Another excellent method for balsam mounts is to heat the slides until the 
balsam is soft and then remove the cover- glasses. The turpentine cleaning mix- 
ture, etc., can then act on the entire surface. It should be said, however, that at 
the present price of slides and cover-glasses it costs nearly as much as the slides 
and covers are worth to clean those that have been used in balsam mounting. 

\ 236. Cover-Glasses or Covering Glasses. — These are circular or quadr- 
angular pieces of thin glass used for covering and protecting microscopic objects. 
They should be very thin, 0.10 to 0.25 millimeter (see table, \ 29). It is better 
never to use a cover-glass over o. 20 mm. thick , then the preparation ma)- be studied 
with a 2 mm. oil immersion as well as with lower objectives. Except for objects 
wholly unsuited for high powers, it is a great mistake to use cover-glasses thicker 
than the working distance of a homogeneous objective (§ 61). Indeed, if one 
wishes to employ high powers, the thicker the sections the thinner should be the 
cover-glass (see \ 240). 

The cover-glass should always be considerably larger than the object over 
which it is placed. 

\ 237. Cleaning Cover-Glasses. — New cover-glasses should be put into a 
glass dish of some kind containing one of the cleaning mixtures (£ 242) and al- 
lowed to remain a da}- or longer. In putting them in, push one in at a time and 
be sure that each is entirely immersed, otherwise they adhere very closely and the 
cleaning mixture is unable to act freely. Soiled covers should be left a week or 
more in the cleaning mixture. An indefinite sojourn in the cleaner does not seem 



CH. VII] 



SLIDES AND COVER-GLASSES 



163 



to injure the slides or covers. After one day or longer, pour off the cleaning mix- 
ture into another glass jar, and rinse the cover-glasses, moving them around with 
a gentle rotary motion. Continue the rinsing until all the cleaning mixture is 
removed. One may rinse them occasionally, and in the meantime allow a very 
gentle stream of water to flow on them, or they may be allowed to stand quietly 
and have the water renewed from time to time. When the cleaning mixture is 
removed rinse the covers well with distilled water, and then cover them with 50% 
to 75% alcohol. 





Figs. 129-130. Figures of square and of circular cover-glasses. See also Fig. 
162 for covers on serial sections. 

\ 238. Wiping the Cover-Glasses. — When read} 7 to wipe the cover-glasses, 
remove several from the alcohol and put them on a soft, dry cloth, or on some of 
the lens or filter paper to let them drain. Grasp a cover-glass by its edges, cover 
the thumb and index finger of the other hand with a soft, clean cloth or some of the 
the lens paper. The bleached cheese cloth ( \ 235 ) is good for wiping covers. Grasp 
the cover between the thumb and index and rub the surfaces. In doing this it is 
necessary to keep the thumb and index well opposed on directly opposite faces of 
the cover so that no strain will come on it, otherwise the cover is liable to be 
broken. 

When a cover is well wiped, hold it up and look through it toward some dark 
object. The cover will be seen partly by transmitted and partly by reflected light, 
and any cloudiness will be easily detected. If the cover does not look clear, breathe 
on the faces and wipe again. If it is not possible to get a cover clear in this way 
it should be put again into the cleaning mixture. 

As the covers are wiped put them in a clean glass box or Petri dish. Handle 
them always by their edges, or use fine forceps. Do not put the fingers on the 
faces of the covers, for that will surely cloud them. 

Fig. 131. Glass dish for holding covers 
( Whitall, Tatuni & Co. ) . 

\ 239. Cleaning Large Cover-Glasses. — For 
serial sections and especially large sections, 
large quadrangular covers are used (Fig. 162). 
These are to be put one by one into a cleaning 
mixture as for the smaller covers and treated in 

every way the same. In wiping them one may proceed as for the small covers, 
but special care is necessary to avoid breaking them. It is especially desirable 
that these large covers should be thin — not over 0.15-0.20 mm. otherwise high ob- 
jectives cannot be used in studying the preparations. 

\ 240. Measuring the Thickness of Cover-Glasses. — It is of the greatest 
advantage to know the exact thickness of the cover-glass on an object ; for, (a) 




164 



SLIDES AND COVER-GLASSES 



\CH. VII 



In studying the preparation one would not try to use objectives of a shorter work- 
ing distance than the thickness of the cover ($61); (b) In using adjustable 
objectives with the collar graduated for different thicknesses of cover, the collar 
might be set at a favorable point without loss of time ; (c) For unadjustable 
objectives the thickness of cover may be selected corresponding to that for which 
the objective was corrected (see table, \ 29). Furthermore, if there is a variation 
from the standard, one ma3' remedy it, in part at least, by lengthening the tube if 
the cover is thinner, and shortening it if the cover is thicker than the standard 
{I 102, Fig. 133.) 

In the so called No. 1 cover-glasses of the dealers in microscopical supplies, 
the writer has found covers varying from o. 10 mm. to 0.35 mm. To use cover- 
glasses of so wide a variation in thickness without knowing whether one has a 
thick or thin one is simply to ignore the fundamental principles by which correct 
microscopic images are obtained. 




Fig. 132. Micrometer Calipers {Brown and Sharpe). Pocket Calipers, gradu- 
ated in inches or millimeters, and well adapted for measuring cover-glasses. 



It is then strongly recommended that every preparation shall be covered with 
a cover-glass whose thickness is known, and that this thickness should be indicated 
in some way on the preparation. 

\ 241. Cover-Glass Measurers, Testers or Gauges. For the purpose of 
measuring cover-glasses there are three very excellent pieces of apparatus. The 
micrometer calipers (Fig. 132) used chiefly in the mechanic arts, are convenient 
and from their size easily carried in the pocket. The two cover-glass measurers 
specially designed for the purpose are shown in Figs. 133-134. With either of 
these the covers may be more rapidly measured than with the calipers. 

With all of these measures or gauges one should be certain that the index 
stands at zero when at rest. If the index does not stand at zero it should be 
adjusted to that point, otherwise the readings will not be correct. 

As the covers are measured the different thicknesses should be put into 
different boxes and properly labeled. Unless one is striving for the most accurate 
possible results, cover-glasses not varying more than 0.06 mm. may be put in the 
same box. For example, if one takes 0.15 mm. as a standard, covers varying 0.03 
mm. on each side may be put into the same box. In this case the box would con- 
tain covers of 0.12, 0.13, 0.14, 0.15, 0.16, 0.17 and 0.1S mm. 



CH. VII] 



SLIDES AND COVER-GLASSES 



165 




Fig. 133. Cover-Glass Measurer {Edward Bausch). 

The cover glass is placed in the notch between the two screws, and the drum is 
turned by the milled head at the right till the cover is in contact with the screws. 
The thickness is then indicated by the knife edge on the drum and may be read off 
directly in 0.001 of a millimeter or inch. In other columns is given the proper 
tube-length for various unadjustable objectives (J, \, \, and ^ in.) {Bausch and 
Lomb Optical Company). 




Fig. 134. Zeiss Cover-Glass 
Measurer. With this the knife edge 
jaws are opened by means of a lever 
and the cover inserted. The thick- 
ness may then be read off on the face 
as the pointer indicates the thick- 
ness in hundredths millimeter in 
the outer circle and in thousandths 
inch on the inner circle. 



\ 242. Cleaning Mixtures for Glass. — The cleaning mixtures used for clean- 
ing slides and cover-glasses are those commonly used in chemical laboratories : 
(Dr. G. C. Caldwell's Laboratory Guide in Chemistry). 



1 66 MOUNTING AND LABELING [CH. VII 

(A) Dichromate of Potash and Sulphuric Acid. 

Dichromate of potash (K 2 Cr 2 7 ) - - 200 grams 

Water, distilled or ordinary - - - 800 cc. 

Sulphuric acid (H 2 S0 4 ) - 1200 cc. 

Dissolve the dichrornate in the water by the aid of heat, using an agate or 
other metal dish, then pour it into a heavy iron kettle lined with sheet lead (Pr. 
Trans. Amer. Micr. Soc, 1899, p. 107). Add the sulphuric acid to the dissolved 
dichromate in the kettle. The purpose of the lead lined kettle is to avoid break- 
age from the great heat developed upon the addition of the sulphuric acid. The 
lead is very slightly affected by the acid, iron would be corroded by it. 

For making this mixture, ordinary water, commercial dichromate and strong 
commercial sulphuric acid may be used. It is not necessary to employ chemically 
pure materials. 

This is an excellent cleaning mixture and is practically odorless. It is exceed- 
ingly corrosive and must be kept in glass vessels. It may be used more than 
once, but when the color changes markedly from that seen in the fresh mixture it 
should be thrown away. 

( B) Sulphuric and Nitric Acid Mixture. 

Nitric acid (HN0 3 ) - - 200 cc. 

Sulphuric acid (H 2 S0 4 ) _____ 300 cc. 

The acids should be strong, but they need not be chemically pure. The two 
acids are mixed slowly, and kept in a glass stoppered bottle. This is a more cor- 
rosive mixture than (A), and has the undesirable feature of giving off stifling 
fumes, therefore it must be carefully covered. It may be used several times. It 
acts more rapidly than the dichromate mixture, but on account of the fumes is not 
so well adapted for general laboratories. 

MOUNTING, AND PERMANENT PREPARATION OF MICROSCOPICAL OBJECTS 

\ 243. Mounting a Microscopical Object is so arranging it upon some suit- 
able support (glass slide) and in some suitable mounting medium that it may be 
satisfactorily studied with the microscope. 

The cover-glass on a permanent preparation should always be considerably 
larger than the object ; and where several objects are put under one cover-glass it is 
false economy to crowd them too closely together. 

\ 244. Temporary Mounting. — In a great many cases objects do not need to 
be preserved ; they are then mounted in any way to enable one best to study 
them, and after the study the cover glass is removed, the slide cleaned and made 
ready for future use. In the study of living objects, of course only temporary 
preparations are possible. With amoebae, white blood corpuscles, and many 
other objects both animal and vegetable, the living phenomena can best be studied 
by mounting them in the natural medium. That is, for amoebae, in the water in 
which they are found ; for the white blood corpuscles, a drop of blood is used and, 
as the blood soon coagulates, they are in the serum. Sometimes it is not easy or 
convenient to get the natural medium, then some liquid that has been found to 
serve in place of the natural medium is used. For many things, water with a 



CH. VII] 



MOUNTING AND LABELING 



167 



little common salt (water 100 cc, common salt y^ths gram) is employed. This is 
the so-called normal salt or saline solution. For the ciliated cells from frogs and 
other amphibia, nothing has been found so good as human spittle. Whatever is 
used, the object is put on the middle of the slide and a drop of the mounting 
medium added, and then the cover-glass. The cover is best put 
on with fine forceps, as shown in Fig. 136. After the cover is in 
place, if the preparation is to be studied for some time, it is better 
to avoid currents and evaporation by painting a ring of castor oil 
around the cover in such a way that part of the ring will be on the 
slide and part on the cover (Fig. 165. ) 



Fig. 135. Needle Holder {Queen & Co.). By means of the 
screw clamp or chuck at one end the needle may be quickly 
changed. 

Fig. 136. To show the method of putting a cover-glass upon a 
microscopic preparation. The cover is grasped by o?ie edge, the 
opposite edge is then brought down to the slide, and the cover 
gradually lowered upon the object. 




Fig. 136. 



\ 245. Permanent Mounting. — For making permanent microscopical prepara- 
tions, there are three great methods. Special methods of procedure are necessary 
to mount objects successfully in each of these ways. The best mounting medium 
and the best method of mounting in a given case can only be determined by ex- 
periment. In most cases some previous observer has already made the necessary 
experiments and furnished the desired information. 

The three methods are the following : ( A) Dry or in air ( § 246); (B) In some 
medium miscible with water, as glycerin or glycerin jelly {\ 250); (C) In some 
resinous medium like Canada Balsam (f 255). 

\ 246. Mounting Dry or in Air.— The object should be thoroughly dry. If 
any moisture remains it is liable to cloud the cover-glass, and the specimen may 
deteriorate. As the specimen must be sealed, it is necessary to prepare a cell 
slightly deeper than the object is thick. This is to support the cover-glass, and 
also to prevent the running in by capillarity of the sealing mixture. 

\ 246a. Order of Procedure in Mounting Objects Dry or in Air. 

1. A cell of some kind is prepared. It should be slightly deeper than the 
object is thick {\ 248). 

2. The object is thoroughly dried (dessicated) either in dry air or by the aid 
of gentle heat. 

3. If practicable the object is mounted on the cover-glass ; if not it is placed 
in the bottom of the cell. 

4. The slide is warmed till the cement forming the cell wall is somewhat 
sticky, or a very thin coat of fresh cement is added ; the cover is warmed and put 



i68 



MOUNTING AND LABELING 



\_CH. VII 



on the cell and pressed down all around till a shining ring indicates its adherence 

(l 249)- 

5. The cover-glass is sealed (§ 249). 

6. The slide is labeled ( \ 308 ) . 

7. The preparation is cataloged and safely stored (§ 309, 311). 

\ 247. Example of Mounting Dry, or in Air. — Prepare a shallow cell and dry 
it (§ 24S). Select a clean cover-glass slightly larger than the cell. Pour upon the 
cover a drop of \o% solution of salycilic acid in 95% alcohol. Let it dry spon- 
taneously. Warm the slide till the cement ring or cell is somewhat sticky, then 
warm the cover gently and put it on the cell, crystals down. Press on the cover 
all around the edge (? 246); seal, label and catalog (§ 253, 308, 309). 

A preparation of mammalian red blood corpuscles may be satisfactorily made 
by spreading a very thin layer of fresh blood on a cover with the end of a slide. 
After it is dry, warm gently to remove the last traces of moisture and mount blood 
side down, precisely as for the crystals. One can get the blood as directed for the 
Micro-spectroscopic work [\ 210). 




Fig. 137. Turn-Table for sealing cover-glasses and making shallow mount- 
ing cells. ( Queen & Co.) 

\ 248. Preparation of Mouuting Cells. — (A) Thin Cells. These are most 
conveniently made of some of the cements used in microscopy. Shellac is one of 
the best and most generally applicable. To prepare a shellac cell place the slide 
on a turn-table (Fig. 137) and center it, that is, get the center of the slide over 
the center of the turn-table. Select a guide ring on the turn-table which is a little 
smaller than the cover-glass to be used, take the brush from the shellac, being 
sure that there is not enough cement adhering to it to drop. Whirl the turn-table 
and hold the brush lightly on the slide just over the guide ring selected. An 
even ring of the cement should result. If it is uneven, the cement is too thick or 
too thin, or too much was on the brush. After a ring is thus prepared remove 
the slide and allow the cement to dry spontaneously, or heat the slide in some 
way. Before the slide is used for mounting, the cement should be so dry when it 
is cold that it does not dent when the finger nail is applied to it. 

A cell of considerable depth may be made with the shellac by adding succes- 
sive layers as the previous one drys. 

(B) Deep Cells are sometimes made by building up cement cells, but more 
frequently, paper, wax, glass, hard rubber, or some metal is used for the main 
part of the cell. Paper rings, block tin or lead rings are easily cut out with gun 
punches. These rings are fastened to the slide by using some cement like the 
shellac. 



CH. VII] 



MOUNTING AND LABELING 



169 



\ 249. Sealing the Cover-Glass for Dry Objects Mounted in Cells. — When 
an object is mounted in a cell, the slide is warmed until the cement is slightly 
sticky or a very thin coat of fresh cement is put on. The cover-glass is warmed 
slightly also, both to make it stick to the cell more easily, and to expel any re- 
maining moisture from the object. When the cover is put on it is pressed down 
all around over the cell until a shining ring appears, showing that there is an in- 
timate contact. In doing this use the convex part of the fine forceps or some 
other blunt, smooth object ; it is also necessary to avoid pressing on the cover 
except immediately over the wall of the cell for fear of breaking the cover. When 
the cover is in contact with the wall of cement all around, the slide shonld be 
placed on the turn-table and carefully arranged so that the cover-glass and cell 
wall will be concentric with the guide rings of the turn-table. Then the turn- 
table is whirled and a ring of fresh cement it painted, half on the cover and half 
on the cell wall (Fig. 165.) If the cover-glass is not in contact with the cell wall 
at any point and the cell is shallow, there will be great danger of the fresh cement 
running into the cell and injuring or spoiling the preparation. When the cover- 
glass is properly sealed, the preparation is put in a safe place for the drying of the 
cement. It is advisable to add a fresh coat of cement occasional!}-. 



mi 







Fig. 138. Centering Card. A card with stops for the slide and circles in the 
position occupied by the center of the slide. If the slide is put upon such a card it 
is very easy to arrange the object so that it will be approximately in the center oj 
the slide. The position of the long cover used for serial sections is also shown 
{Fig. 162). (From the Microscope, December, 1886). 

I 250. Mounting Objects in Media Miscible with Water. — Many objects are 
so greatly modified by drying that they must be mounted in some medium other 
than air. In some cases water with something in solution is used. Glycerin of 
various strengths, and glycerin jelly are also much employed. All these media 
keep the object moist and therefore in a condition resembling the natural one. 
The object is usually and properly treated with gradually increasing strengths of 
glycerin or fixed by some fixing agent before being permanently mounted in 
strong glycerin or either of the other media. 



170 MOUNTING AND LABELING [CH. VII 

In all of these different methods, unless glycerin of increasing strengths has 
been used to prepare the tissue, the fixing agent is washed away with water before 
the object is finally and permanently mounted in either of the media. 

For glycerin jelly no cell is necessary unless the object has a considerable 
thickness. 

\ 251. Order of Procedure in Mounting Objects in Glycerin. 

1. A cell must be prepared on the slide if the object is of considerable thick- 
ness (g 248, 249). 

2. A suitably prepared object ( \ 250) is placed on the center of a clean slide, 
and if no cell is required a centering card is used to facilitate the centering 
(Fig- 138). 

3. A drop of pure glycerin is put upon the object, or if a cell is used, enough 
to fill the cell. 

4. In putting on the cover-glass it is grasped with fine forceps and the under 
side breathed on to slightly moisten it so that the glycerin will adhere, then one 
edge of the cover is put on the cell or slide and the cover gradually lowered upon 
the object (Fig. 136). The cover is then gently pressed down. If a cell is used, a 
a fresh coat of cement is added before mounting ($ 249). 

Fig. 139. Slide and cover-glass showing method of 
anchoring a cover-glass with a glycerin preparation when 
no cell is used. A cover-glass so anchored is not liable to 
move when the cover is being sealed ($ 253). 




*©<] 



Fig. 140. Glass slide with cover-glass, a drop of re- 
agent arid a bit of absorbent paper to show method of irri- 
gation (I 262, 263). 



5. The cover-glass is sealed (§ 249). 

6. The slide is labeled ( § 30S). 

7. The preparation is cataloged and safely stored (J 309, 311). 

\ 252. Order of Procedure in Mounting Objects in Glycerin Jelly. 

1. Unless the object is quite thick no cell is necessary with glycerin jelly. 

2. A slide is gently warmed and placed on the centering card (Fig. 138) and 
a drop of warmed glycerin jelly is put on its center. The suitably prepared object 
is then arranged in the center of the slide. 

3. A drop of the warm glycerin jelly is then put on the object, or if a cell is 
used it is filled with the medium. 

4. The cover-glass is grasped with fine forceps, the lower side breathed on 
and then gradually lowered upon the object (Fig. 136) and gently pressed down. 

5. After mounting, the preparation is left flat in some cool place till the 
glycerin jelly sets, then the superfluous amount is scraped and wiped away and 
the cover-glass sealed with shellac (? 253). 

6. The slide is labeled (§ 30S). 

7. The preparation is cataloged and safely stored ($ 309, 311 ). 

I 253, Sealing the Cover-Glass when no Cell is used. — (A) For glycerin 
mounted specimens. The superfluous glycerin is wiped away as carefully as possi- 
ble with a moist cloth, then four minute drops of cement are placed at the edge of 
the cover (Fig. 139), and allowed to harden for half an hour or more. These will 



CH. VW] 



MOUNTING AND LABELING 



171 



anchor the cover-glass, then the preparation may be put on the turn-table and a 
ring of cement put around the edge while whirling the turn-table. 





Fig. 141. A — Simple form of moist chamber made with a plate and bowl. B, 
bowl serving as a bell jar ; P, plate containing the water and over which the bowl is 
inverted ; S, slides on which are mounted preparations which are to be kept moist. 
These slides are seen endwise and rest upon a bench made by cementing short pieces 
of large glass tubing to a strip of glass of the desired length and width. 

B — Two cover -glasses (C) made eccentric, so that they may be more easily sepa- 
rated by grasping the projecting edge. 

C — Slide (S) with projecting cover-glass (C). The projection of the cover en- 
ables one to grasp and raise it without danger of moving it on the slide and thus 
folding the substance under the cover. (From Proc. Amer. Micr. Soc. , 1891 ). 

(B) For objects in glycerin jelly, Earranls' solution or a resinous medium. 
The mounting medium is first allowed to harden, then the superfluous medium is 
scraped away as much as possible with a knife, and then removed with a cloth 
moistened with water for the glycerin jelly and Farrants' solution or with alcohol, 
chloroform or turpentine, etc., if a resinous medium is used. Then the slide is put 
on a turn-table and a ring of the shellac cement added. (C) Balsam preparations 
may be sealed with shellac as soon as they are prepared, but it is better to allow 
them to dry for a few days. One should never use a cement for sealing prepara- 
tions in balsam or other resinous media if the solvent of the cement is a solvent 
also of the balsam, etc. Otherwise the cement will soften the balsam and finally 
run in and mix with it, and partly or wholly ruin the preparation. Shellac is 
an excellent cement for sealing balsam perparations, as it never runs in. Balsam 
preparations are rarely sealed. 

§ 254. Example of Mounting in Glycerin Jelly. — For this select some stained 
and isolated muscular fibres or other suitably prepared objects. (See under isola- 
tion \ 259). Arrange them on the middle of a slide, using the centering card, and 
mount in glycerin jelly as directed in § 252. Air bubbles are not easily removed 
from glycerin jelly preparations, so care should be taken to avoid them. 

\ 255. Mounting Objects in Resinous Media. — While the media miscible 
with water offer many advantages for mounting animal and vegetable tissues the 
preparations so made are liable to deteriorate. In many cases, also, they do not 
produce sufficient transparency to enable one to use high enough powers for the 
demonstration of minute details. 



172 MOUNTING AND LABELING [CH. VII 

By using sufficient care almost any tissue may be mounted in a resinous 
medium and retain all its details of structure. 

For the successful mounting of an object in a resinous medium it must in 
some way be deprived of all water and all liquids not miscible with the resinous 
mounting medium. There are two methods of bringing this about : (A) By dry- 
ing or desiccation {\ 256), and (B) by successive displacements (§ 258). 




Fig. 142. Small spirit lamp modified into a balsam 
bottle, a glycerin or glycerin -jelly bottle, or a bottle 
for homogeneous immersion liquid. For all of these 
purposes it should contain a glass rod as shown in the 
figure. By adding a small brush, it answers well for 
a shellac bottle also {See Fig. 170). 



\ 256. Order of Procedure in Mounting Objects in Resinous Media by 
Desiccation : 

1. The object suitable for the purpose (fly's wings, etc. ) is thoroughly dried 
in dry air or by gentle heat. 

2. The object is arranged as desired in the center of a clean slide on the 
centering card (Fig. 138). 

3. A drop of the mounting medium is put directly upon the object or spread 
on a cover-glass. 

4. The cover-glass is put on the specimen with fine forceps (Fig. 136), but in 
no case does one breathe on the cover as when media-miscible with water are 
used. 

5. The cover-glass is pressed down gentl} 7 . 

6. The slide is labeled {\ 308). 

7. The preparation is cataloged and safely stored {\ 309, 311 ). 

I 257. Example of Mounting in Balsam by Desiccation. — Find a fresh fly, 
or if in winter, procure a dead one from a window sill or a spider's web. 
Remove the fly's wings, being especially careful to keep them the dorsal side 
up. With a camel's hair brush remove any dirt that may be clinging to them. 
Place a clean slide on the centering card, then with fine forceps put the two wings 
within one of the guide rings. Leave one dorsal side up, turn the other ventral 
side up. Spread some Canada balsam on the face of the cover-glass and with the 
fine forceps place the cover upon the wings (Fig. 136). Probably some air-bubbles 
will appear in the preparation, but if the slide is put in a warm place these will 
soon disappear. Label, catalog, etc., (1307-311). 

$ 25S. Mounting in Resinous Media by a Series of Displacements. — For ex- 
amples of this see the procedure in the paraffin and in the collodion methods 



CH. VII] ISOLATION OF ELEMENTS 173 

(| 280, 300). The first step in the series is Dehydration, that is, the water is dis- 
placed by some liquid which is miscible both with the water and the next liquid 
to be used. Strong alcohol (95% or stronger) is usually employed for this. Plenty 
of it must be used to displace the last trace of water. The tissue may be soaked 
in a dish of the alcohol, or alcohol from a pipette may be poured upon it. Dehy- 
dration usually occurs in the thin objects to be mounted in balsam in 5 to 15 min- 
utes. If a dish of alcohol is used it must not be used too many times, as it loses 
in strength. 

The second step is clearing. That is, some liquid which is miscible with the 
alcohol and also with the resinous medium is used. This liquid is highly refrac- 
tive in most cases, and consequently this step is called clearing and the liquid a 
clearer. The clearer displaces the alcohol, and renders the object more or less 
translucent. In case the water was not all removed, a cloudiness will appear in 
parts or over the whole of the preparation. In this case the preparation must be 
returned to alcohol to complete the dehydration. 

One can tell when a specimen is properly cleared by holding it over some 
dark object. If it is cleared it can be seen only with difficulty, as but little light 
is reflected from it. If it is held toward the window, however, it will appear 
translucent. 

The third and final step is the displacement of the clearer by the resinous 
mounting medium. 

The specimen is drained of clearer and allowed to stand for a short time till 
there appears the first sign of dullness from evaporation of the clearer from the 
surface. Then a drop of the resinous medium is put on the object, and finally a 
cover-glass is placed over it, or a drop of the mounting medium is spread on the 
cover and it is then put on the object. 

ISOLATION OF HISTOLOGICAL ELEMENTS 

\ 259. For a correct conception of the forms of the cells and fibers of the 
various organs of the body, one must see these elements isolated and thus be able 
to inspect them from all sides. It frequently occurs also that the isolation is not 
quite complete, and one can see in the clearest manner the relations of the cells 
or fibers to one another. 

The chemical agents or solutions for isolating are, in general, the same as 
those used for hardening and fixing. But the solutions are only about one-tenth 
as strong as for fixing, and the action is very much shorter, that is, from one or 
two hours to as many days. In the weak solution the cell cement or connective 
tissue is softened so that the cells and fibers may be separated from one another, 
and at the same time the cells are preserved. In fixing and hardening, on the 
other hand, the cell cement, like the other parts of the tissue, are made firmer. 
In preparing the isolating solutions it is better to dilute the fixing agents with 
normal salt solution [\ 331) than merely with water. 

\ 260. Isolation by Means of Formaldehyde. — Formaldehyde in normal 
salt solution is one of the very best dissociating agents for brain tissue and all the 
forms of epithelium. It is prepared as follows : 2 cc. of formal, (that is, a 40% 
solution of formaldehyde) are mixed with 1000 cc. of normal salt solution. This 
acts quickly and preserves delicate structures like the cilia of ordinary epithelia, 



174 



I SO LA TION OF ELEMENTS 



[CH. VII 



and also of the endymal cells of the brain. It is satisfactory for isolating the 
nerve cells of the brain. For the epithelium of the trachea, intestines, etc., the 
action is sufficient in two hours ; good preparations may also be obtained after 
two days or more. The action on nerve tissue of the brain and myel or spinal 
cord is about as rapid. For the stratified epithelia, like those of the skin, mouth, 
etc. , it may require two or three days for the most satisfactory preparations. 





ooo 


ooo 


ooo 


ooo 


ooo 



Fig. 143 A. Fig. 143 B. Fig. 144. 

Fig. r43. Preparation Vials for Histology and Embryology. This repre- 
sents the two vials, natural size, that have been found most useful. They are kept 
in blocks with holes of the proper size. 

Fig. 144. Block with holes for containing shell vials. 

\ 261. Example of Isolation. — Place a piece of the trachea of a very recently 
killed animal, or the roof of a frog's mouth, in the formaldehyde dissociator. 
After two hours or more, up to two or three days, excellent preparations of ciliated 
cells may be obtained by scraping the trachea or roof of the mouth and mounting 
the scrapings on a slide. If one proceeds after two hours, probably most of the 
cells will cling together, and in the various clumps will appear cells on end show- 
ing the cilia or the bases of the cells, and other clumps will show the cells in pro- 
file. By tapping the cover gently with a needle holder or other light object the 
cells will be more separated from one another, and many fully isolated cells will 
be seen. 

\ 262. Staining the Cells. — Almost any stain may be used for the formalin 
dissociated cells. For example, one may use eosin. This may be drawn under 
the cover of the already mounted preparation (Fig. 140), or a new preparation may 
be made and the scrapings mixed with a drop of the eosin before putting on the 
cover-glass. It is an advantage to study unstained preparations, otherwise one 
may obtain the erroneous opinion that the structure cannot be seen unless it is 



CH. VII] 



ISOLATION OF ELEMENTS 



175 



stained. The stain makes the structural features somewhat plainer ; it also accen- 
tuates some features and does not affect others so markedly. 

I 263. Permanent Preparations of Isolated Cells.— If one desires to make a 
permanent preparation of isolated cells it may be done by placing a drop of glycerin 
at the edge of the cover and allowing it to diffuse under the cover, or the diffusion 
may be hurried by using a piece of blotting paper, as shown in Fig. 140. One 
may also make a new preparation and either with or without staining mix the 
cells with a drop of glycerin on the slide and then cover, or one may use glycerin 
jelly (\ 254, 326). 




Fig. 145. Adjustable lens holder with universal joint. This is especially use- 
ful J vt gross dissections, and for dissecting the partly isolated elements with needles 
(Leitz: Wm. Krafft, N. Y.). 

\ 264. Isolation of Musculer Fibers. — For this the formal dissociator may be 
used {\ 260, 324), but the nitric acid method is more successful (§ 330). The fresh 
muscle is placed in this in a glass vessel. At the ordinary temperature of a sitting 
room (20 degrees centigrade) the connective tissue will be so far gelatinized in 
from one to three days that it is easy to separate the fascicles and fibers either with 
needles or by shaking in a test tube or reagent vial (Fig. 143) with water. It 
takes longer for some muscles to dissociate than others, even at the same temper- 
ture, so one must try occasionally to see if the action is sufficient. When it is, the 
acid is poured off and the muscles washed gently with water to remove the acid. 
If one is ready to make the preparations at once they may be isolated and mounted 
in water. If it is desired to keep the specimen indefinitely or several days, the 
water should be poured off and a half saturated solution of alum added ($ 314). 
The alum solution is also advantageous if the specimens are to be stained. The 



176 



COLLODION SECTIONING 



[CH. VII 



specimens may be mounted in glycerin, glycerin jelly or balsam. Glycerin jelly 
is the most satisfactory, however. 




Fig. 146. Pfeiffer 's preparation microscope with erecting prism between the 
objective and ocular {Leiiz ; Wm. Krafft, New York). 

THE PREPARATION OF SECTIONS OF TISSUES AND ORGANS 



$ 265. At the present time there are three principal methods of obtaining 
thin sections of tissues and organs for microscopic study. These methods are : 
The Collodion Method, the Paraffin Method, and the Freezing Method. Each of 
these methods has its special application, although the collodion method is per- 
haps the most generally applicable, and the freezing method the most restricted, 
and is used mostly in pathological work where rapid diagnosis is necessary and 
the finest details of structure are not so important. With the paraffin method the 
thinnest sections may be made, and in some ways it is the most satisfactory of all. 
A good microtome is of great aid in sectioning. 

\ 266. The Collodion Method. — In sectioning by this method the tissues are 
first hardened properly and then entirely infiltrated with collodion, and the collod- 
ion hardened. It is not removed from the tissue, since on account of its transpar- 
ency it does no harm. 

\ 267. Fixing and Hardening the Tissue. — Any of the approved methods 
of hardening and fixing may be employed. A good general method which is 
applicable to nearly all of the tissues and organs is that by Picric- Alcohol. For 
the preparation of the solution see (§ 333). A small piece of tissue or organ not 



CH. VII '] COLLODION SECTIONING 177 

containing more than two to three cubic centimeters is placed in 40 or 50 cc. of 
the picric-alcohol and left 6 to 24 hours, when the first picric-alcohol should be 
thrown away and fresh added. After one or two days more the picric- 
alcohol should be poured off and 67% alcohol added. In a day or two this is re- 
placed by 75% or 82% alcohol ; 82% is on the whole most satisfactory, and the 
tissue may be left in this till it is ready for dehydration. 

\ 26S. Dehydration before Infiltration. — When one is ready to imbed for 
sectioning, the tissue must first be dehydrated in plenty of 95% or stronger alcohol. 
It is better to take only a small piece for this. The smaller the piece the thinner 
the sections that may be made. The dehydration will usually be completed in 2 to 
24 hours. If the alcohol is changed two or three times the dehydration will be 
hastened. 

\ 269. Saturating with Ether- Alcohol. (f 322). — The next step is to remove 
the tissue from the alcohol and place it in a vial of ether-alcohol (J 322) for 2 to 
24 hours. The dehydration becomes somewhat more complete by this step, and 
the tissue is more perfectly prepared for the reception of the collodion. If the 
dehydration is very thorough in the alcohol, this step may be ommitted, however, 
but one is surer of success if the ether-alcohol is used. 

\ 270. Infiltration with Thin Collodion. — The ether-alcohol is poured off, 
and a mixture of thin collodion is added (§ 319). Two or three hours will suffice 
for objects two or three millimeters in thickness. A stay of one or more days 
does no harm. The larger the object the more time is needed. 

\ 271. Infiltration with Thick Collodion. — The thin collodion is poured off 
and thick collodion {\ 319) added. For very small objects, four or five hours 
will suffice to infiltrate, but for larger objects a longer time is necessary. The 
tissue does not seem to be injured at all in the thick collodion, and a stay in it 
of a day or even a week is more certain to insure a perfect infiltration. 

§ 272. Imbedding. — The tissue may be imbedded in a paper box, such as is 
used for paraffin imbedding, or in any of the other boxes devised for paraffin. It 
is better, if paper is used, to put a small amount of oil on the paper to prevent 
the collodion from sticking to it. Vaselin spread over lightly and then re- 
moved, so far as possible, with a cloth or with lens paper, gives the right surface. 
For small objects it is more convenient to imbed immediately on a holder that 
may be clamped into the microtome. Cylinders or blocks of glass, vulcanite, 
wood and cork have all been recommended and used. A cork of the proper size 
is most convenient, and for many purposes answers well. Some collodion is put 
on the end of the cork and a pin put near one edge. The tissue is transferred 
from the thick collodion to the cork and leaned against the pin. Drops of the 
thick collodion are then poured on the tissue, and by moving the cork properly 
the thick viscid mass may be made to surround and envelop the tissue. Drops 
of collodion are added at short intervals until the tissue is well surrounded, and 
then as soon as a slight film hardens on the surface, the cork bearing the tissue is 
inverted in a wide-mouth vial of considerably larger diameter than the cork (Fig. 
143). The vial should contain sufficient chlorform to float the cork. The vial is 
then tightly corked. In imbedding somewhat larger objects on the end of a cork 
or other holder, it is frequently advantageous to wind oiled paper around the 
holder or cork, tie it tightly and have the projecting hollow cylinder sufficiently 
long to receive the object. The tissue is then put into the cylinder and sufficient 



I7§ COLLODION SECTIONING \CH. VII 

collodion added to completely immerse it. As soon as a film has formed over the 
exposed end, the cork may be inverted and immersed in chloroform as described 
above. For the use of "deck plugs" see \ 274. 

I 273. Hardening and Clarifying the Collodion. — After a few hours the 
collodion is hardened by the chloroform. If it acts long enough, and if no water 
is present, the imbedding mass is rendered entirely transparent. Whenever the 
collodion is hard, whether it is clear or not, the chloroform is poured off and the 
castor-xylene* clarifier (g 317) added. In a few hours the imbedded mass will be- 
come as transparent as glass and the tissue will seem to have nothing around it. 
The tissue may remain for years in the castor-xylene. Sometimes the collodion 
remains white and opaque for a considerable time. So far as the writer has been 
able to judge, this is due to moisture. If one breathes on the mass too much while 
imbedding, or if it is very damp in the room, opacity may result. Sometimes, in 
objects of considerable size, this may remain for a week. This is the exception, 
however, and if the mass seems sufficiently hard and tough, the cutting may pro- 
ceed even if the clarification is incomplete, t 

In case the imbedding mass will not clarify after a few days the imbedded 
object may be placed in 95% alcohol for a day for dehydration, and then passed 
through chloroform and into the clarifier. There is usually no trouble in getting 
the mass perfectly clear in this way. 

If one is in a great hurry, the collodion may be hardened in 10 or 15 minutes 
by heating the bottle containing the chloroform in a water bath. The imbedding 
block of hardened collodion may then be transferred to the castor-xylene clarifier 
and kept warm. It will soon clear the collodion. One can then cut the sections. 

§ 274. Cutting the Sections. — For cutting the sections the collodion block is 
usually fastened to some form of holder. For small objects cork is fairly good. 
Blocks of glass, vitrified fiber, etc., have been used. If one uses wood the "deck 
plugs" of the shipwright are satisfactory. They are about the right length when 
one plug is made into two holders by sawing in two (Ewing & Ferguson). To 
fasten the collodion block to any form of holder, remove it from the castor-xylene, 
trim as desired, then dry the end on blotting paper, pour some thick collodion on 
the holder and press the collodion block down into the collodion. The evapora- 
tion usually fixes it in two or three minutes, when the holder may be clamped in 
the jaws of the microtome and the cutting proceed. For collodion sectioning a 
long, drawing cut is necessary in order to obtain thin, perfect sections. The ob- 
ject is, therefore, put in the jaws of the microtome at the right level, and the 
knife arranged so that half or more of the blade of the knife is used in cutting the 
section. It is advantageous also to have the object with its long diameter parallel 
with the edge of the knife. The surrounding collodion mass should be cut away, 
as in sharpening a lead pencil, so that there is not more than a thickness of about 



*The hydrocarbon xylene (C 8 H 10 ) is called xylol in German. In English, 
members of the hydrocarbon series have the termination "ene," while members 
of the alcohol series terminate in "ol. " 

t The imbedded object may remain in the castor-xylene clarifier indefinitely 
without harm. The collodion grows somewhat tougher by a prolonged stay in it. 
After cutting all the sections desired at one time, the imbedded tissue is returned 
to the clarifier for future sections. 



CH. VII] 



COILODION SECTIONING 



179 



two millimeters all around the tissue. This is to render the diameter of the end to 
be cut as small as possible. The smaller the object the thinner can the sections be 
made. With an object two or three millimeters thick and not over five milli- 
meters wide, and a good sharp knife, sections 5// to 6/c can be cut without diffi- 
culty. When knife and tissue are properly arranged the tissue and the knife are 
flooded with the clarifier. Make the sections with a steady motion of the knife. 
Then draw the section up toward the back of the knife with an artist's brush and 
make the next section. Arrange the sections in serial order on the knife blade 
till enough are cut to fill the area that the cover-glass will cover. For large objects 
one can cut thinner sections by a kind of sawing cut. 

\ 275, Transferring the Sections to the Slide. — If the clarifier has evaporated 
so as to leave the sections somewhat dry on the knife, add a small amount. Take 
a piece of thin absorbent, close-meshed paper about twice the size of a slide and 
place it directly upon the sections. Press the paper down evenly all around and 
then pull the paper off the edge of the knife. The sections will adhere to the 
paper. Place the paper, sections down, on a slide, taking care that the sections 
are in the desired position on the slide. Use some ordinary lens paper or some 
absorbent paper, and press it down gently upon the transfer paper. This will ab- 
sorb the oil, and then the transfer paper may be lifted, with a rolling motion, 
from the slide. The sections will remain on the slide. (See notes p. 180). 

\ 276. Fastening the Sections to the. Slide. — Drop just enough ether- 
alcohol (equal parts of sulphuric ether and 95% alchol) on the sections to moisten 
them. This will melt the collodion and fasten the sections to the slide. Allow the 
slide to remain in the air till the surface begins to look slightly dull or glazed. 

Sometimes, especially when the air is moist, the sections wrinkle badly when 
the ether-alcohol is put on to fasten them to the slide. The excessive wrinkling 
can be avoided by using one part alcohol and two parts ether instead of using 
equal parts of each. Perhaps also it would be advantageous in this case to use 
absolute alcohol. 




Fig. 147. Reagent bottle with combined cork and pipette {This 
is made by taking a cork of the proper size and making in it a hole 
with a cork borer for the glass tube. It is advantageous to have a 
string tied tightly around the rubber bulb as shown). 

\ 277. Removing the Oil from the Sections. — As soon as the 
ether-alcohol has evaporated sufficiently to leave the surface dull, 
place the slide in a jar of ordinary commercial benzin. It may be 
left here a day or more without injury to the sections, but if 
moved around in the jar the oil will be removed in three to five 
minutes. From the benzin transfer to a jar of 95Q-0 alcohol to 
wash away the benzin. One may use alcohol in the beginning, 
but it dissolves the oil far less rapidly than the benzin. The slide 
may remain in the alcohol half a day or more if one wishes, but a 
stay of five minutes or a thorough rinsing of half a minute or so 
by moving the slide around in the alcohol will suffice. 

Xylene is to be preferred to benzin for removing the oil, but 
it is more expensive. 



Fig. 147. 



180 COLLODION SECTIONING \CH. VII 

\ 278. Staining the Sections with an Alcoholic Stain. — If an alcoholic 
stain containing 50% or more alcohol (for example, hydrochloric acid carmine in 
70% alcohol) is used, the slide may be removed from the 95% alcohol, drained 
somewhat and then the stain poured upon the sections, or preferably, the slide 
immersed in a jar of the stain. The stain is finally washed away with 67% or 
stronger alcohol, the sections dehydrated in 95% alcohol, cleared and mounted in 
balsam. 

I 279. Staining the Sections with an Aqueous Dye. — In staining with a 
watery stain, the slide bearing the sections is transferred from the 95% alcohol 
and plunged into a jar of water, and either allowed to remain a few minutes or 
moved around in the water a moment. Then it is placed horizontally and some 
of the stain placed on the sections with a pipette, or preferably, it is immersed in 
a jar of the stain ; in case of immersion the slide should stand vertically or nearly 
so, then any particles of dust, etc., in the stain will settle to the bottom of the 
vessel and not settle on the sections. When the sections are stained, usually 
within five minutes, they are thoroughly washed with water either by the use of a 
pipette or preferably by immersing in a jar of water. They may then be counter- 
stained for half a minute with some general dye, like eosin or picric acid, or 
mounted with but the one stain, j 



^Various forms of paper have been used to handle the collodion sections. It 
should be moderately strong, fine meshed and not liable to shed lint, and fairly 
absorbent. One of the first and most successful papers recommended is "closet or 
toilet paper. " Cigarette paper is also excellent. In my own work the heavy white 
tissue paper has been found almost perfect for the purpose. Ordinary lens paper 
or thin blotting paper for absorbing the oil may be used with it. ($ 275). 

tlf one is a long time in cutting a series of sections, it sometimes occurs that 
the xylene evaporates, and while the sections may not look dry, they are practically 
in castor oil and not easily transferable. In such a case fresh clarifier or even a 
little xylene to thin the oil on the sections may be used. If the oil is too thick it 
is viscid and there is difficulty in handling the sections with the paper as they 
stick rather firmly to the knife. (§275). 

fin the past the plan for changing sections from 95% alcohol to water, for ex- 
ample, has been to run them down gradually, using 75, 50 and 35% alcohol, suc- 
cessive!}'. Each percentage may vary, but the principle of a gradual passing from 
strong alcohol to water was advocated. On the other hand I have found that the 
safest method is to plunge the slide directly into water from the 95% alcohol. The 
diffusion currents are almost or quite avoided in this way. There is no time for 
the alcohol and water to mix, the alcohol is washed away almost instantly by the 
flood of water. So in dehydrating after the use of watery stains, the slide is 
plunged quickly into a jar of 95% alcohol. The diffusion currents are avoided in 
the same way, for the water is removed by the flood of alcohol. This plan has 
been submitted to the severe test of laboratory work, and has proved itself perfectly 
satisfactory. 



CH. VII] 



COLLODION SECTIONING 





Fig. 148. 



Fig. 150. 



Fig. 148. Waste bowl with rack for supporting slides and a small funnel in 
which the slides stand while draining. This outfit is easily made by any tinsmith. 
The rack is composed of two brass rods about 4 mm. in diameter. The bent end 
pieces are sheet lead. The funnel is made of tin, copper or brass. Either copper 
or brass is preferable to tin. A glass dish like that shown in Fig. 160 is better than 
a bowl, as it can be more readily and thoroughly cleaned. {Cut loaned by Wm. 
Wood & Co. ) 

Fig. 149. Round glass aquarium. This glass vessel is better than the bowl 
for all the uses described for the bowl. ( Whitall, Tatum & Co. ) 

Fig. 150. Glass box or ointment jar with cover. These boxes may be had of 
various sizes and can be used advantageously Jor water, and for cleaning mixture 
for slides and cover glasses (\ 242). ( Whitall, Tatum & Co. ) 




Fig. 151. Section lifter. This is of thin, springy, flexible metal placed in a 
handle as shown. These are made of various sizes for large or small sections. Such 
an instrument is exceedingly helpful in handling loose sections. (Queen & Co.) 



182 



COLLODION SECTIONING 



[CH. VII 





Fig. 152. Perforated sectio m lifter. This is easily made by soldering a wire 
to some very thin sheet brass or copper, and then perforating this with a coarse 
needle or fine awl. Any roughness must be removed by using a fine oil stone. 

ORDER OF PROCEDURE IN MAKING MICROSCOPICAL PREPARATIONS BY THE 

COLLODION METHOD 
\ 280. It will be seen from this table, and sections 267-281, that it requires 
about five days to get a microscopical preparation if one commences with the 
fresh tissue. Other methods of hardening might require as many months. It is 
evident, therefore, that one must exercise foresight in histology or much time 
will be wasted. 

1. Fixing and hardening the tissues 12. Removing the oil from the sections 



(§ 267), 4 days or more. 

2. Dehydrating the object to be cut in 
95% or stronger alcohol (§ 268), 2- 
24 hours. 

3. Saturating the tissue in ether-alcohol 
(§ 269), 2-24 hours. 

4. Infiltrating with thin collodion 
($ 270), 2 hours to 2 days. 

5. Infiltrating in thick collodion ( § 27 1 ) , 
5 hours to several days. 

6. Imbedding the tissue (§272), 15 to 
20 minutes. 

7. Hardening the collodion with chlo- 
roform {I 273), 5-24 hours. 

8. Clarifying and further hardening the 
collodion with castor-xylene (§ 273), 
10-36 hours. 

9. Cutting the sections (§ 274), 10 min- 
utes to 2 hours. 

10. Transferring the sections to a slide 
with paper (§ 275), 1 minute. 

11. Fastening the sections to the slide 
with ether-alcohol (§ 276), 1 or 2 
minutes. 



with benzin and alcohol (§ 277), 3-5 
minutes, or 24 hours. 

13. Staining the sections with an alco- 
holic dye (§ 278), 2 minutes to 24 
hours. 

14. Staining the sections with an aque- 
ous dye (§ 279), 2-10 minutes. 

15. Removing the superfluous dye by 
washing in water or alcohol (§ 278- 
279), 2-5 minutes. 

16. Staining with a general dye (§ 279), 
15-30 seconds. 

17. Washing with water or alcohol 
(§ 278-279), 1 to 2 minutes. 

18. Dehydrating the sections in 95% al- 
cohol (§281), 5 min. to 24 hours. 

19. Clearing the sections (§281 ), 5 min- 
utes to 24 hours. 

20. Draining the sections, 1-2 minutes. 

21. Mounting in Canada balsam ($ 281), 
1-2 minutes. 

22. Labeling the preparation ($ 308), 2 
minutes. 

23. Cataloging the preparation ($ 309), 
5-10 minutes. 



CH. VII] PARAFFIN SECTIONING 183 

\ 281. Mounting in Balsam. — After the sections are stained they must be 
dehydrated and cleared before mounting in balsam. For the dehydratiou the slide 
is plunged into a jar of 95% alcohol. For clearing after the dehydration the slide 
is drained of alcohol and put down flat and the clearer poured on, or the whole 
slide is immersed in a jar of clearer (\ 318). Clearing usually is sufficient in a 
few minutes ; a stay of an hour or even over night does not injure most sections. 

In mounting in balsam the clearer is drained away by standing the slide nearly 
vertically on some blotting paper, or by using the waste bowl and standing it up 
in the little funnel (Fig. 148). Then the balsam is put on the sections or spread 
on the cover-glass and that placed over the sections. 

For cataloging and labeling, see $ 307-310. 



Fig. 153. Small spirit lamp modified into a bal- 
sam bottle, or a glycerin or glycerin-jelly bottle, or a 
bottle for homogeneous immersion liquid. For all of 
these purposes it should contain a glass rod. See also 
Fig. 16S. 




\ 2S2. The Collodion Method with Alcohol.— A good method of procedure 
for making collodion sections is to do exactly as described, including \ 272, and 
then instead of hardening the collodion in chloroform and clarifier, it is hardened 
in 82% alcohol for a day or two before sectioning. In sectioning the knife and 
tissue are kept wet with 82% alcohol and the sections are dehydrated with 95% 
alcohol and then fastened to the slide with ether alone or with ether-alcohol. 
The staining and mounting (? 278-281) are as described. One may preserve the 
tissue after imbedding for a long time in the 82% alcohol before sectioning and 
sections may be made at any time. While this method appears somewhat simpler, 
the results are not so satisfactory as by the oil method given above. 

THE PARAFFIN METHOD 

§ 283. As with the collodion method, the tissues are first properly fixed and 
hardened and then entirely filled with the imbedding mass, but unlike the collo- 
dion the mass must be entirely removed before the sections are finally mounted. 
The tissue thus imbedded and infiltrated is like a homogeneous mass and sections 
may be cut of extreme thinness. 

\ 284. Harden perfectly fresh tissue in picric-alcohol ($ 333) from one to 
three days. (Any good method for fixing and hardening the elements may be 
used. One must observe in each case, however, the special conditions necessary 



PARAFFIN SECTIONING 



[CH. VII 



for each method. The time might be longer or shorter than for the picric-alcohol. 
(See Lee, the Microtomists' Vade-Mecum. ) 

If picric-alcohol is used, pour it off after the proper time for fixing has 
elapsed, and add 67% alcohol. Leave this on the tissue from one to three days, 
and if it becomes very yellow it is well to change it two or three times. After two 
or three days pour off the 67% alcohol and add 82%. The tissue should remain 
in this one or two days, and it maj^ remain indefinitely. 

In case the alcohol becomes much yellowed, it should be changed. 

I 285. Dehydration and Preparation for Imbedding. — From the pieces of 
tissue fixed and hardened in any approved manner, cut pieces 5 to 10 millimeters 
long and 2 to 3 millimeters in breadth. Place one or two pieces in a shell vial 
(Fig. 143.) and add 95% alcohol. Change the alcohol after two or three hours, 
and within 6 to 24 hours, depending on the size of the piece to be dehydrated, the 
dehydration will be completed. The secret of success is the use of plenty of 
alcohol and sufficient time. Absolute alcohol for the second change would act 
more promptly and efficiently, but if plenty of 95% is used one will succeed, 
unless the day, or the climate in general, is too damp. 




Fig. 154. Copper pail with 
water bottom for melting para- 
ffin. This also serves as a water 
bath for large bottles in which 
saturated solutions of dichro- 
mate and other salts are pre- 
pared. 



(If one is studying organs, then the whole organ may need to be prepared for 
imbedding, but for minute structure small pieces are preferable, as thinner 
sections may be made. ) 

\ 286. Displacing Alcohol and Clearing Tissues with Cedar-wood Oil and 
Infiltrating with Paraffin. — (Lee, p. 66. Neelson and Schiefferdecker, Arch, fiir 
Anat. und Physiol., 1SS2, p. 206.) When the tissue is dehydrated it is removed 
to a vial of cedar-wood oil. When the alcohol used for dehydration is displaced 
by the oil, the tissue will look clear and translucent. This requires 2 to 24 hours. 



CH. VII] 



PARAFFIN SECTIONING 



185 



It is hastened by warmth. It is then removed from the cedar-wood oil, drained, 
and placed in pure, melted paraffin, and this is then put into a paraffin oven and 
left from 2 to 24 hours (see Ch. X). It is then imbedded for sectioning.* 

Paraffin for infiltrating has usually a somewhat lower melting point than that 
for imbedding. Equal parts of paraffin of 43 C. and 54 C, answer well. For 
imbedding, the paraffin must be of a melting point which will give good ribbons 
in the temperature of the room where the sectioning is to be done. In a room of 
19 to 20 C. a mixture of 1 part 43 C. paraffin with two parts of 54 C. usually 
answers well. 



Fig. 155. Hot filter for 
paraffin, gelatin, balsam, etc. 
It is entirely surrounded by a 
water jacket. The water is 
heated by placing a burner imder 
the projecting part H. The 
wire basket is to hold the strainer 
and allow a free flow of the 
filtered substance on all sides. 
There is a bail for suspending 
the filter, and the filtered sub- 
stance rims out through the 
narrowed part F. 




\ 287. Imbedding in Paraffin. — Make a small paper box, fill it nearly full 
of hot paraffin, place the box for a half minute on some cold water to make a 
thin solid layer on the bottom, then transfer the tissue to the box and arrange 
near one end so that the sections may be cut in the desired plane. Now pi ace the 
paper box on as cold water as possible so that the paraffin may cool quickly. It 
will be more homogeneous if cooled quickly, and will shrink tightly against the 
tissue and avoid air spaces. 

In imbedding two main things should be looked after : The paraffin should 
be hot so that it will thoroughly fuse with the paraffin in the tissue. The tissue 
should be kept from the bottom of the box, either by holding it up in the middle 
of the box with warmed forceps or a perforated section lifter while a stratum 



*Thickened cedar-wood oil like that for oil immersion objectives is recom- 
mended by Lee for clearing. This is very expensive, and for most work unneces- 
sary. Any form of cedar-wood oil has been found satisfactory in the writer's 
laboratory. The great thing is to have the tissue thoroughly dehydrated before 
putting it into the oil. 



iS6 



PARAFFIN SECTIONING 



\CH. VII 



cools on the bottom, or a stratum of the paraffin may be cooled on the bottom 
before putting the tissue in the box. Cool quickly after the tissue is in place. 




FiG. 156. Paraffin re- 
ceptacle P, with water 
bath and spout for par- 
affin imbedding(/S8j ) . 



I 288. Cutting the Sections.— After the imbedding mass is well cooled, re- 
move the paper box and trim the end containing the tissue in a pyramidal form, 
clamp the block of paraffin in the holder of the microtome so that the tissue will be 
at the proper level for cutting. If a ribbon microtome is used, heat the holder and 
melt the end of the block upon it. Cool and place the holder in its place in the 
microtome. Use a very sharp, dry razor for cutting the sections. The sections 
are made with a rapid, straight cut as in planing. Do not try to section with a 
drawing cut as in collodion sectioning. If the temperature of the room is right 
for the paraffin used, the sections will remain flat, and if the opposite sides of the 
block are parallel, and one edge strikes the knife squarely, the sections will adhere 
and thus make a ribbon. If the room is too cold for the paraffin the sections 
will roll. If it is too warm the sections will crumple. 

Remember the sections must be very thin, from 3/* to 15// to show fine struc- 
tural details to good advantage. 

The secret of making good ribbons of sections is to have the block of paraffin 
containing the tissue cut square and properly arranged in the microtome so that 
the block strikes the edge of the section knife at right angles with the edge ; and 
finally the paraffin must be of a proper melting point for the room in which the 
sections are to be cut. Remember that the larger the object the thicker must 
be the sections, and the softer the paraffin. Frequently one may modify the tem- 
perature if too cold by a Bunsen burner flame near the microtome. If it is too 
warm one may go to a basement room. 

$ 289. Extending Sections with Warm Water. — Paraffin sections are liable 
to have fine wrinkles or folds in them. These folds are very annoying and often 
obscure the structure. To get rid of them the sections are extended or stretched 
upon warm water. One may put a ribbon of sections on warm water and then cut 
the ribbon into pieces and transfer the pieces to slides. Practically, however, the 



CH. VII} 



PARAFFIN SECTIONING 



187 



extension is almost always accomplished on the slide itself. A slide is albumen- 
ized ('i 290) and the ribbon cut into short pieces and placed on the slide. Distilled 
or filtered water is then added with a pipette (Fig. 147) until the sections float. 
Then the slide is moved back and forth over an alcohol or gas flame to warm the 
water. Care must be taken to avoid melting the paraffin. As the water warms 
the paraffin containing the sections will flatten and stretch out. One will be sur- 
prised at the amount of extension. It is necessary to take pieces considerably 
shorter than the cover-glass to be used or when extended the sections will not all 
be covered. After the sections are extended, arrange the ribbons carefully on the 
slide as shown in Fig. 162 if one is making serial sections. Arrange in the middle 
of the slide if only one or two sections are on each slide (Fig. 138). Let the ex- 
cess water drain off. Now let the slide stand several hours for the water to evapor- 
ate completely. The time will depend on the temperature and the dryness of the 
atmosphere. If there is plenty of time, leave the slides 24 or 48 hours. If one 
has a register with hot air intake, the slides may be put in the current of hot air. 
They will dry out in half an hour or an hour. Sections which have been left for a 
vear have given excellent results. 





Fig. 157. Trays for slides and for ribbons of sections. The figures show the 
construction. It is important to have the bordering f tame with rounded corners so 
that the trays may be easily pulled out of a pile or reinserted. The screw eye shown 
in A makes it easy to pull out a single tray. For ribbons of sections a piece of paper 
is placed in the tray and the ribbons are placed on it. (A) Face view, (B) Sec- 
tional view of the whole tray, (C) Sectional view of one side to show the construc- 
tion more clearly. Trays of this kind are so cheap {$15.00 pet hundred for those 
holding 50 to 60 slides), that a laboratory can have a great number. (Trans. Amer. 
Micr, Soc, 1899, p. 107). 

The slide trays (Fig. 157) are excellent for drying preparations of all kinds. 
\ 290. Fastening the Sections to the Slide. — To fasten the sections firmly 
to the slide, coat the slide with albumen fixative (§ 312) as follows : Put a minute 



PARAFFIN SECTIONING 



[CH. VII 



drop of the albumen on the center of a slide and with a clean finger spread the 
albumen over the slide, wiping off all that is possible. Finally beat or tap the 
slide with the end of the finger. This will make a very thin (it cannot be too 
thin) and even layer. 

The sections are extended and dried as described in \ 289. When the sections 
are thoroughly dry they are in optical contact with the slide and have a shining 
appearance when looking on the back of the slide. When the sections are dry 
coat them with }4% collodion made as follows. Take % gram of soluble cotton, 
put it into a bottle and add 60 cc. of 95% or absolute alcohol, and 40 cc. of sul- 
phuric ether. Coat the sections with a soft camel's hair brush. The collodion 
should dry in a minute or less. If one uses too much ether in this fixing collo- 
dion, the paraffin will be partly dissolved and will look moist for a long time. 
The slight excess of alcohol will obviate anv such difficultv. 





Fig. 158. A slide holder and 
bottle for containing the same {Mix, 
Journal of Applied Microscopy, vol. 
1, 1898, p. 169.) 



Fig. 759. Slide holder with the 
bail hinged so that it may be turned 
aside in inserting or removing the 
slides. 



When the collodion is dry place the slide in benzin or xylene to dissolve 
the paraffin (see \ 291). If the sections are not extended on water, they may be 
put directly on the albumenized sides, pressed down with the finger and coated 
with collodion. This is much more rapid, but does not get rid of the fine folds. 
(See Dr. Agnes Claypole Trans. Amer. Micro. Soc. 1894, p. 66, 127.) 

\ 291. Removing the Paraffin. — Immerse the slide in a vessel of xylene or 
benzin. This will dissolve the paraffin. An hour will usually suffice. One can 



CH. VII] 



PARAFFIN SECTIONING 



hasten the solution of the paraffin by moving the slide in the solvent. In this way 
it may be dissolved in 5 to 10 minutes, or even less. It will do no harm to leave 
the slide in the benzin or xylene over night. Two or three days even might not 
do any harm, but it is usually better to proceed at once to the other operations. 




Fig. 160. Apparatus and regents with which the slide holders are used. With 
this apparatus it is easy to prepare specimens in large numbers very expeditiously. 
After the sectiofis are fastened to the slide and placed in the holder, the slides need 
not be touched during all the operations until they are finally ready to be mounted 
in balsam. Each holder contains from 1 2 to 1 4. slides. (§ 2jy-joo). The bottles 
for the reagents are glass stoppered specimen or museum bottles. {Mix, four. Ap. 
Micr. /SgS, p. 171. ) Compare Fig. 148. 



\ 292. Removing the Xylene or Benzin — From the xylene or benzin plunge 
the slide bearing the sections into a jar of 95% alcohol, and leave it for a few 
minutes, or move it around in the alcohol for half a minute or so. 

\ 293. Staining the Sections with an Alcoholic Dye. — With an alcoholic 
stain like hydrochloric acid carmine, remove the slide from the alcohol, and add 
the stain directly after draining the slide, Do not allow the stain to become dry, 
for that would injure the tissue. Wash away the stain with 67% alcohol, then 
dehydrate with 95% alcohol, clear and mount in balsam as described below. 

\ 294. Staining with as Aqueous Dye. — Wash away the 95% alcohol from 
the slide bearing the sections by plunging it into a jar of water and moving it 
around a moment. Then add the stain to the sections with a pipette, or immerse 
the slide in a jar of the stain, and allow the stain to act from 5 to 10 minutes. 
Wash thoroughly with water. 

\ 295. Staining with a General Dye — Counterstaining. — If it is desired to 
give a general stain after the nuclear dye ($ 294), carmine stained preparations 
may be tinted with picric-alcohol for half a minute or more ( \ 333), and the hem- 
atoxylin stained specimens with eosin ( \ 321 ). It usually takes less than a minute 
for this. Wash away the counterstain with water. 



190 



PARAFFIN SECTIONING 



\CH. VII 



\ 296. Counterstaining with Picro-fuchsin. —For a general dye to use with 
hematoxylin, eosin is good, but to differentiate the tissues more completely, 
especially connective tissue, which is present in practically every section made, it 
is better to use Van Gieson's mixture of picric acid and acid fuchsin. (Picric 
acid, saturated aqueous solution 75 cc, water 25 cc. 1% aqueous solution of acid 
fuchsin, 10 cc.) Sections are first strongly stained with hematoxylin, well washed 
with water and then stained 3 seconds to 15 minutes in the picro-fuchsin. They 
are then washed in distilled water ; or in tap water, to which has been added a drop 
or two of glacial acetic acid to 100 cc. of water. They are then dehydrated, 
cleared and mounted in acid balsam, that is in balsam which has not been neutral- 
ized ($ 315). If glycerin or glycerin jelly is used as a mounting medium it should 
be slightly acid. Unless the mounting medium is slightly acid, the red of the 
acid fuchsin soon fades. In some cases less acid fuchsin should be used, and in 
some a greater amount. Acid fuchsin alone without the picric acid is also good 
for a counterstain. The picro-fuchsin is a very valuable differential stain and 
combined in different proportions with picric acid will give great assistance in al- 
most every case. It does not seem to be a permanent stain. (See Freeborn, 
Trans. N. Y. Path, Soc, 1S93, p. 73. Also studies from the department of pathol- 
ogy of the College of Physicians and Surgeons, Columbia University, N. Y., 
1 894- 1 895). 

161. Copliu's staining dish. A. The 
entire dish; B. the dish in cross section. 
This is made of glass and is a very neat piece 
of apparatus. With it ten slides may be 
stained at once. ( Whitall, Tatum & Co. ) 

\ 297. Dehydration of the Stained Sec- 
tions. — Place the slide with the stained sec- 
dons in a jar of 95% or absolute alcohol and 
save it a few minutes, or wave it around in 
the alcohol for half a minute or so. 

Remember that the larger and thicker 

the sections the more time it requires to de- 

cross-section hydrate them. In a moist atmosphere, 95% 

SHOWING SLIDES / . ^ y0/0 

in position. alcohol is not completely satistactory, and 
one must use a stronger alcohol. When it is dry, 95% answers very well. 

\ 298. Clearing the Sections. — Drain off the alcohol, and place the slide in a 
jar of clearer (§ 318, A or B) or put a drop or two of clearer on the sections. The 
clearing is usually accomplished in two or three minutes. 

\ 299. Mounting in Balsam. — For this the clearer is drained from the 
slide, and wiped away with blotting paper, cloth, etc. The balsam is then put 
upon the sections and the cover added, or a cover-glass is spread with the balsam 
and then put over the sections. ( If the sections show a whitish appearance and 
are opaque they were not sufficiently dehydrated. If natural balsam is used the 
sections will clear up in time). 




CH. VII] 



SERIAI SECTIONS 



191 



ORDER OF PROCEDURE IN MAKING MICROSCOPICAL PREPARATIONS BY THE 

PARAFFIN METHOD. 



# 300. It will be seen from this table and from sections 283 to 299 that it re- 
quires from 5 to 7 days to get a microscopical preparation by the paraffin method 
if one starts with a fresh tissue. Depending on the method of fixing and harden- 
ing, the time may be much greater. Much time will be lost in waiting unless one 
plans ahead in histological work. 



Fixing and hardening the tissue or 

organ (§ 284), 4 days or more. 
Dehydrating the object to be cut in 

95% or stronger alcohol ($ 285), 

1 to 24 hours. 
Displacing the alcohol and clearing 

tissues with cedar-wood oil. (See 

I 286), 2 to 24 hours. 
Infiltrating the tissue with paraffin 

in the paraffin oven (§ 286), 2 to 

24 hours. 
Imbedding in paraffin ($ 287), 10 

minutes. 
Cutting the sections (| 288), 10 min- 
utes. 
Extending the sections with warm 

Water. (See \ 289.) 
Fastening the sections to a slide 

{\ 290), 5 minutes to 24 hours. 
Removing the paraffin (£291), 10 

minutes to 24 hours. 
Removing the xylene or benzin 

(3 292). 



11. Washing with water, note, p. 180. 

12. Staining with an aqueous dye 

($ 294), 2 minutes to 24 hours. 

13. Washing away the superfluous stain 

with water {\ 294).) 

14. Staining with a general dye (§ 295- 

296), 10 seconds to 10 minutes. 

15. Washing the sections with water 

{\ 295-296). 

16. Dehydrating the stained sections in 

95% alcohol {\ 297), 3 minutes to 
24 hours. 

17. Clearing the sections (§ 298) 2 min- 

utes to 24 hours. 

18. Mounting in Balsam {\ 299), 1 to 5 

minutes. 

19. Sealing the cover-glass (\ 253), 2 

minutes. 

20. Labeling the preparation ($ 30S), 2 

minutes. 

21. Cataloging the preparation (§ 309), 

5 to to minntes. 



SERIAL SECTIONS 



\ 301. In histological studies it is frequently of the greatest advantage to 
have the sections in serial order, then an obscure feature in one section is fre- 
quently made clear by the following or preceding sections. While serial sections 
are very desirable in histological study, they are absolutely necessary for the solu- 
tion of morphological problems presented in complex organs like the brain, in 
embryos and in minute animals where gross dissection is impossible. 

\ 302. Arrangement of Tissues for Sections in Histology. — They should be so 
arranged that the exact relations of each part to the organ can be readily deter- 
mined. For example, an organ like the intestine, a muscle or a nerve, should be 
so arranged that exact transections or longisections can be made. Organs like the 
liver and other glands, the skin, etc., should be so arranged that sections parallel 
with the surface or at right angles to it, (surface or vertical sections) maybe 
made. Oblique sections are often very puzzling. 



192 SERIAL SECTIONS [CH. VII 

With cylindrical objects, especially botanical specimens, one may cut tangen- 
tial sections, i. e., sections at right angles to a radius, or parallel with the radii 
(radial sections), or transections, i. e., sections across the long axis. 

§ 303. Arrangement of Serial Sections. — The numerical order may be very 
conveniently like the words on a printed page, i. e., beginning at the upper left 
hand corner and extending from left to right. 

The position of the various aspects of the sections should be in general such 
that when they are under the compound microscope the rights and lefts will corre- 
spond with those of the observer. This may be accomplished as follows for sec- 
tions made in the three cardinal sectional planes, Transections, Frontal Sections, 
Sagittal Sections : 

(C) Transection, i. e., sections across the long axis of the embryo or animal 
dividing it into equal or unequal cephalic and caudal parts. 

(a) In accordance with the generally approved method of numbering serial 
parts in anatomy, the most cephalic section should be first (Fig. 162). 

(b ) The caudal aspect of the section should face upward toward the cover- 
glass, the cephalic aspect being next the slide. 

(c) The dorsal aspect should face toward the upper edge of the slide 
(Fig. 162). 

This arrangement may be easily accomplished for the transections as follows : 
Imbed the embryo with the right side down, taking the precautions against letting 
the embryo rest against the bottom of the box ($ 287). 

(1) Mount the block of paraffin containing the embryo so that the tail end 
will be next the microtome holder. The head will then be cut first. 

(2) Place in the microtome so that the right side of the embryo will meet the 
edge of the knife. 

(3) Mount as a printed line and the first or cephalic section will be at the 
upper left hand corner, and the dorsal aspect of the embryo will be toward the 
upper edge of the slide. 

Under the microscope the rights and lefts will appear as in the observers own 
body, also the dorsal and ventral aspects so that he can easily locate parts by 
comparing them with his own body. 

(B) Frontal Sections, i. e., sections lengthwise of the embryo or animal and 
from right to left (dextral and sinistral), so that it is divided into equal or unequal 
dorsal and ventral parts. 

Imbed the embryo with the right side down in the imbedding box as before. 

( 1 ) Mount the paraffin block so that the ventral side of the embryo is next 
the microtome holder. The dorsal side will then be cut first. 

(2) Let the right side of the embr}-o meet the edge of the knife. 

(3) Mount the first section on the left end of the slide as before and so that 
the sections will be crosswise on the slide, the tail toward the upper edge. Under 
the compound microscope the head will appear toward the upper edge and the 
rights and lefts will be as in the observer's own body. 

(C) Sagittal Sections, that is sections lengthwise of the embrj^o or animal 
and from the ventral to the dorsal side, thus dividing the body into equal or 
unequal right and left parts. 

For these sections imbed the embryo as in the two preceding cases. 



CH. VI1~\ 



SERIAL SECTIONS 



193 



(1) Put the right side of the embryo next the microtome holder, then the 
left side will be cut first. 

(2) Let the caudal end meet the knife edge. 

(3) Put the first section in the upper left hand part of the slide as in the 
other cases. The sections will be lengthwise of the slide. This will bring the 
ventral side up and the head to the right on the slide. Under the microscope the 
head will appear at the left and the dorsal side toward the upper edge of the slide. 

I 304. For serial sections with collodion imbedded objects it is a great advan- 
tage to have the imbedding mass unsymmetrically trimmed, so that if a section is 
accidentally turned over it may be easily noticed and rectified. 

Furthermore it is imperatively necessary that the object be so imbedded that 
the cardinal aspects, dextral and sinistral, dorsal and ventral, cephalic and caudal, 
shall be known with certainty. 

I 305. Thickness of Cover-Glass and of Serial Sections. — It is a great advan- 
tage to use very thin cover-glasses ( 0.12-0. 18 mm.) for serial sections, then the 
cover will not prevent the use of high powers. When the ordinary slides (25 X 
76 mm., 1X3 inch) are used cover-glasses 24 X 50 mm. may be advantageously 
employed. 



79 


1 


jHj|»:£JBtt''VjpHlS , Ji 


\o 


Horn© 5 


5 
10 
86 


o I 


t& oil 





Slide 10 
See's 79 


T. 
93 


o| 


SESEaHl 


D 


20m 100 
1901 






Fig. 162. 







Fig. 162. A slide of serial sections showing the position of the sections, the 
label and the size of the cover-glass (24 x 50 mm. ). The numbers next the cover 
at the left indicate the first section of each row, and illustrate the method of placing 
the sections as if they were wordsr on a printed page. . T. Transections. 



The combined thickness of the sections on a slide is easily determined by not- 
ing carefully the position of the microtome screw at the first and last sections and 
measuring the elevation. With good modern automatic microtomes the successive 
sections are almost exactly uniform in thickness, hence it is easy to determine the 
combined thickness of the sections on a slide. 

\ 306. Labeling Serial Sections. — The label of a slide on which serial sections 
are mounted should contain at least the following : 

The name of the embryo and the number of the series ; the number of the 
slide of that series ; the thickness of the sections, and the number of the first and 
last section on the slide ; the date. It is also a convenience to have the informa- 
tion repeated in part on the left end and the number of the first section in each 
row, as shown in the sample slide of serial sections. (Fig. 162). 



194 



LABELING AND CATALOGING 



{CH. VII 



LABELING, CATALOGING AND STORING MICROSCOPICAL PREPARATIONS 

\ 307. Every person possessing a microscopical preparation is interested in 
its proper management ; but it is especially to the teacher and investigator that 
the labeling, cataloging and storing of microscopical preparations are of import- 
ance. ' ' To the investigator, his specimens are the most precious of his possessions, 
for they contain the facts which he tries to interpret, and the}' remain the same 
while his knowledge, and hence his power of interpretation, increase. They thus 
form the basis of further or more correct knowledge ; but in order to be safe 
guides for the student, teacher, or investigator, it seems to the writer that every 
preparation should possess two things : viz., a label and a catalog or history. This 
•catalog should indicate all that is known of a specimen at the time of its prepara- 
tion, and all of the processes by which it is treated. It is only by the possession 
•of such a complete knowledge of the entire history of a preparation that one is 
able to judge with certainty of the comparative excellence of methods, and thus 
to discard or improve those which are defective. The teacher, as well as the 
investigator, should have this information in an accessible form, so that not only 
he, but his students can obtain at an}' time, all necessary information concerning 
the preparations which serve him as illustrations and them as examples." 

\ 30S. Labeling Ordinary Microscopical Preparations. — The label should 
possess at least the following information (see \ 306 for serial sections): 

EXAMPLE 



(1) The number of the preparation, the 

thickness of the cover-glass and 
of the sections under it. 

(2) The name and source of the prepara- 

tion. 

(3) The date of the specimen ( 2 of 

catalog.) 



No. 475. 



C. AS 

Sees. 8 ju 



Striated Muscle ; transection of the 
Sartorius of the Cat. 

October 15, 1894. 



s./<7 



c< ; l 



vtr 



DATE. QttM 



■2- 



Fig. 163. Example of a label of an ordinary histologic 
specimen. {See also Fig. 162 for serial sections). 



m 



Fig. 163. 



\ 309. Cataloging Preparations. — It is believed from personal experience, and 
from the experience of others, that -each preparation (each slide or each series) 
should be accompanied by a catalog containing at least the information suggested 
in the following formula. This formula is very flexible, so that the order may be 
changed, and numbers not applicable in a given case may be omitted. With many 
objects, especially embryos and small animals, the time of fixing and hardening 
may be months or even years earlier than the time of imbedding. So, too, an 
object may be sectioned a long time after it was imbedded, and finally the sections 



CH. VII] 



LABELING AND CATALOGING 



195 



may not be mounted at the time they are cut. It would be well in such cases to 
give the date of fixing under 2, and under 5, 6 and 8, the dates at which the oper- 
ations were performed if they differ from the original date and from one another. 
In brief, the more that is known about a preparation the greater its value. 



General Formula for Cataloging Mi- 
croscopical Preparations : 

1. The general name and source. 
Thickness of cover glass and of section. 



the 
specimen 



The number of the preparation and 
date of obtaining and fixing the 
the name of the preparator. 



3. The special name of the prepara- 
tion and the common and scientific 
name of the object from which it is de- 
rived. Purpose of the preparation. 

4. The age and condition of the ob- 
ject from which the preparation is de- 
rived. Condition of rest or activity ; 
fasting or full fed at the time of death. 

5. The chemical treatment, — the 
method of fixing, hardening, dissociat- 
ing, etc., and the time required. 

6. The mechanical treatment, — im- 
bedded, sectioned, dissected with nee- 
dles, etc. Date at which done. 

7. The staining agent or agents and 
the time required for staining. 

8. Dehydrating and clearing agent, 
mounting medium, cement used for 
sealing. 

9. The objectives and other accesso- 
ries (micro-spectroscope, polarizer, etc.,) 
for studying the preparation. 

10. Remarks, including references to 
original papers, or to good figures and 
descriptions in books. 



A Catalog Card Written According to 
this Formula : 

Muscular Fibers. Cat. 

C. 15. 
Fibers 20 to 40 u thick. 

2. No. 475. (Drr. IX) Oct. 1, 1891. S. 
H. G., Preparator. 

3. Tendinous and intra-muscular ter- 
minations of striated muscular fibres 
from the Sartorius of the cat (Felts do- 
mestica). 

4 . Cat eight months old, healthy and 
well nourished. Fasting and quiet for 
12 hours. 

5. Muscle pinned on cork with vas- 
elined pins and placed in 20 per cent, 
nitric acid immediately after death by 
chloroform. Deft 36 hours in the acid ; 
temperature 20 C. In alum water {% 
sat. aq. sol. ) 1 day. 

6. Fibers separated on the slide with 
needles, Oct. 3. 

7. Stained 5 minutes with Delafield's 
hematoxylin. 

8. Dehydrated with 95 % alcohol 5 
minutes, cleared 5 minutes with carbol- 
turpentine, mounted in xylene balsam ; 
sealed with shellac. 

9. Use a 16 mm. for the general appear- 
ance of the fibers, then a 2 or 3 mm. ob- 
jective for the details of structure. Try 
the micro-polariscope (§ 218). 
10. The nuclei or muscle corpuscles are 
very large and numerous ; many of the 
intra-muscular ends are branched. See 
S. P. Gage, Proc. Amer. Micr. Sci., 1890, 
p. 132 ; Ref. Hand-book Med., Sci., Vol. 
V., p. 59- 

I 310. General Remarks en Catalogs and Labels. — It is especially desirable 
that labels and catalogs shall be written with some imperishable ink. Some form 
of water-proof carbon ink is the most available and satisfactory. The water-proof 
India ink, or the engrossing carbon ink of Higgins, answers well. As purchased, 
the last is too thick for ordinary writing and should be diluted with one-third its 
volume of water and a few drops of strong ammonia added. 



196 LABELING AND CATALOGING \_CH. VII 

If one has a writing diamond it is a good plan to write a label with it on one 
end of the slide. It is best to have the paper label also, as it can be more easily- 
read. 



Fig. 164. Writing diamond for writing numbers and labels on glass slides^ 
cutting cover-glasses, etc. ( Queen and Co. ) 

The author has found stiff cards, 12^ ^7% cm., like those used for cataloging 
books in public libraries, the most desirable form of catalog. A specimen that is 
for any cause discarded has its catalog card destroyed. New cards may then be 
added in alphabetical order as the preparations are made. In fact a catalog on 
cards has all the flexibility and advantages of the slip system of notes (see Wilder 
& Gage, p. 45). 

Some workers prefer a book catalog. Very excellent book catalogs have been 
devised by Ailing and by Ward (Jour. Roy. Micr. Soc, 1887, pp. 173, 348 ; Amer. 
Monthly Micr. Jour., 1S90, p. 91 ; Amer. Micr. Soc. Proc, 1887, p. 233). 

The fourth division has been added as there is coming to be a strong 
belief, practically amounting to a certainty, that there is a different structural 
appearance in many if not all of the tissue elements depending upon the age of 
the animal, upon its condition of rest or fatigue ; and for the cells of the digestive 
organs, whether the animal is fasting or full fed. Indeed as physiological histology 
is recognized as the only true histology, there will be an effort to determine exact 
data concerning the animal from which the tissues are derived. (See Minot, Proc. 
Amer. Assoc. Adv. Science, 1890, pp. 271-289 ; Hodge, on nerve cells in rest and 
fatigue, Jour. Morph.,vol. VII. (1892), pp. 95-168; Jour. Physiol., vol. XVII. , 
pp. 129-134; Gage, The processes of life revealed by the microscope ; a plea for 
physiological histology, Proc. Amer. Micr. Soc, vol. XVII. (1895), pp. 3-29; 
Science, vol. II., Aug. 23, 1895, pp. 209-218. 

CABINET FOR MICROSCOPICAL PREPARATIONS 

§311. While it is desirable that microscopical preparations should be pro- 
perly labeled and cataloged, it is equally important that they should be protected 
from injury. During the last few years several forms of cabinets or slide holders 
have been devised. Some are very cheap and convenient where one has but a few 
slides. For a laboratory or for a private collection where the slides are numerous 
the following characters seem to the writer essential : 

( 1). The cabinet should allow the slides to lie flat, and exclude dust and light. 

(2). Each slide or pair of slides should be in a separate compartment. At 
each end of the compartment should be a groove or bevel, so that upon depressing 
either end of the slide the other may be easily grasped (Fig. 165). It is also 
desirable to have the floor of the compartment grooved so that the slide rests only 
on two edges, thus preventing soiling the slide opposite the object. 

(3). Each compartment or each space sufficient to contain one slide of the 
standard size should be numbered, preferably at each end. If the compartments 
are made of sufficient width to receive two slides, then the double slides so fre- 
quently used in mounting serial sections may be put into the cabinet in any place 
desired. 



CH. VII] 



IABELING AND CATALOGING 



197 



Fig. 165. A part of a cabinet drawer seen 
jrom above. In compartment No. 96 is repre- 
sented a slide^ lying flat. The label of the 
slide and the number of the compartment are — 
so placed that the number of the compartment 
may be seen through the slide. The sealing ~ 
cement is removed at one place to show that in 
sealing the cover-glass, the cement is put 
partly on the cover and partly on the slide 
(I 249, 253). 

B. — This represents a section of the same ~ 
part of the drawer, (a) Slide resting as in 
a. No. 96. The preparation is seen to be above — 
a groove in the floor of the compartment, (b) 
One e?id of the slide is seen to be uplifted by de- ~ 
pressing the other into the bevel. 

(4). The drawers of the cabinet should be 
entirely independent, so that any drawer may 
be partly or wholly removed without disturb- _ 
ing any of the others. 

(5). On the front of each drawer should be — 
the number of the drawer in Roman numer- > ~ 
als, and the number of the first and last com- 
partment in the drawer in Arabic numerals 
(Fig. 166). 



Fig. 166. Cabinet for Mi- 
croscopical Specimens, show- 
ing the method of arrange- 
ment and of numbering the 
drawers and indicating the 
number of the first and last 
compartment in each drawer. 
It is better to have the slides 
on which the drawers rest 
somewhat shorter, then the 
drawer front may be entire 
and not notched as here shown. 
(From. Proc. Amer. Micr. Soc, 




70 




Fig. 165. 




PREPARATION OF REAGENTS 



[CH. VII 




Fig. 167. Slide box for 25 speci- 
mens. These are cheap and convenient 
and may be stood on end like books, 
thus placing the slides in a horizontal 
position. Smaller boxes, i. e.for j, 6 
and 12 slides are also made, and mail- 
ing boxes for a single slide (Bausch & 
Lomb Co.). 



SOME REAGENTS FOR FIXING, MOUNTING, ETC. 

\ 312. Albumen Fixative (Mayer's). — This consists of equal parts of well- 
beaten white of egg and glycerin. To each 50 cc. of this 1 gram of salicylate of 
soda is added to prevent putrefactive changes. This must be carefully filtered. 
For method of use see \ 290. 

\ 313. Alcohol (Ethylic). — Ethyl alcohol is mostly used for histological pur- 
poses. (A) absolute alcohol (i. e., alcohol of T 9 ff 9 g%) is recommended for many 
purposes, but if plenty of 95% alcohol is used it answers every purpose in 
histology, in a dry climate or in a warm, dry room. When it is damp dehydration 
is greatly facilitated by the use of absolute alcohol. 

(B) 82% alcohol made by mixing 5 parts of 95% alcohol with 1 part of water. 

(C) 67% alcohol made by mixing 2 parts of 95% alcohol with 1 part of water. 
Grades of Alcohol. It has been found by careful tests that quite accurate 

percentages of alcohol may be obtained by mixing water and alcohol as follows : 
Pour alcohol into a graduate until the volume of alcohol corresponds to the de- 
sired percentage. Add water until the volume in cubic centimeters corresponds 
to the original percentage of the alcohol used. For example, to get 67% from 
95% alcohol, pour 67 cc. of 95% into a graduate, and add sufficient water to bring 
the volume up to 95 cc. Far 50% alcohol from 75%, put 50 cc. of 75%' alcohol in 
a graduate, add sufficient water to make the volume 75 cc. From the change in 
volume it does not answer to mix given volumes of water and alcohol in these 
cases. In the first case, if one mixed 75 cc. of 95% alcohol and 20 cc. of water the 
resulting mixture would be over 75% ; but if sufficient water is added to bring the 
volume back to the original percentage more than 20 cc. of water is added, that is 
enough more to compenate for the shrinkage, and the result is approximately 
accurate. 

Methyl Alcohol is much cheaper than ethyl, and answers well in most micro- 
scopical processes. It has recently been so carefully refined that the disagreeable 
odor is very little noticeable. 

Synthol. This is said to be a synthetic form of alcohol. It seems to serve the 
purpose of alcohol in histology. Absolute synthol is far cheaper for the private 
worker than absolute ethyl alcohol. 

I 314. Alum Solution. — For muscle dissociated in nitric acid use a satu- 
rated solution (i. e., a solution in which the water holds all the alum it can. If 
one adds an excess so that there will always be some undissolved alum in the ves- 
sel he can be sure the solution is saturated after it has stood a few daj T s. An easy 
way to get a saturated solution is to take 500 cc. of water and add 100 grams of 
alum and heat the water in an agate dish. All the alum will be melted, but on 



CM. VII ] PR EPA RA TION OF RE A GEN TS 199 

cooling a part will crystallize out, leaving- a cold saturated solution). The satu- 
rated solution may be used but, if a half saturated solution is employed, it will 
answer all the purposes. For a half saturated solution take 100 cc. of water, and 
100 cc. of saturated alum water and mix the two. In case preparations are to be 
kept some time in alum water, 2% of chloral hydrate should be added to prevent 
mold. 

\ 315. Balsam, Canada Balsam, Balsam of Fir; Xylene Balsam. — This is 
one of the oldest and most satisfactory of the resinous media used for mounting 
microscopical preparations. 

The natural balsam is most often used, but within the last ten years the belief 
has arisen that it is better to evaporate the balsam and then dissolve it in xylene 
or benzole. It certainly dries out more rapidly if so treated. Natural balsam has 
the advantage of being able to take up a small amount of water so that if sections 
are not quite dehydrated they will clear up after a time. If xylene balsam is used 
the dehydration must be almost complete or the preparation will look cloudy. 

Filtering Balsam. Balsam is now furnished already filtered through filter 
paper. If one wishes to filter it himself a hot filter like that shown in Fig. 155 is 
good. If xylene balsam is used it may be made thin and filtered without heat. 
For filtering balsam and all resinous and gummy materials, the writer has found a 
paper funnel the most satisfactory. It can be used once and then thrown away. 
Such a funnel may be easily made by rolling a sheet of thick writing paper 
in the form of a cone and cementing the paper where it overlaps, or winding a 
string several times around the lower part. Such a funnel is best used in one of 
the rings for holding funnels. 

Fig. 168. Vessel for homogeneous immersion liquid \ '■"!;j| 

[thick cedar-wood oil). This is filled only a little above the v£ 'i'o 

lower end of the inner tube. The oil will not then run out if f '"' """^ 

the vessel is tipped over. For applying the oil there is a wire i ^~1~~^ '' - - -v"' 1 "^ 

loop attached to the upper cork. The side cork is for the pur- \ ( j 

pose of emptying the bottle, and also for the escape of the air ! i | 

when filling it. [The Spencer Lens Co.; see also Zeit. 

wiss. Mikr., 1897, p. 348, and four. Roy. Micr. Soc, 1898, 1,%.. 

P. 238). V ^-4'^'' : 



Natural Balsam. All the samples of balsam tested by the author have been 
found slightly acid. This is an advantage for carmine, and acid fuchsin stains or 
any other acid stain. Also for preparations injected with carmine or Berlin blue. 
In these cases the color would fade or diffuse if the medium were not slightly 
acid. For hematoxylin the acid is detrimental. For example, the slight amount 
of acid in the balsam will cause the delicate stain in the finest fibers of Weigert 
preparations to fade. Also the fuchsin and other stains which are faded by acids. 
To neutralize the balsam add some pure sodium carbonate, set the balsam in a 
warm place and shake it occassionally. After a month or so the soda will settle 
and the clear supernatant balsam will be found very slightly alkaline. Use this 
whenever an acid medium would fade the stain in the specimen. 



200 PREPARATION OF REAGENTS \CH. VII 

\ 316. Cedar- Wood Oil. — This is used for oil immersion objectives. It is 
best kept in a bottle like that shown in Fig. 168. This oil is quite thick. 

For penetrating tissues and preparing them for infiltration with paraffin, thick 
oil is recommended by Lee. The writer has found, however, that any good cedar 
wood oil gives excellent results in ordinary histological and embryological work. 
That known as Cedar Wood Oil (Florida) is excellent, also that known as cedv 
wood oil (true Lebanon). These forms are far less expensive than that used for 
immersion objectives. The tissues should be thoroughly dehydrated before put- 
ting them into cedar-wood oil, and they should remain until thev are transparent 

{'i 286). 

\ 317. Clarifier, Castor-Xylene Clarifier. — This is composed of castor oil 1 
part and xylene* 3 parts. (Trans. Amer. Micr. Soc, 1S95, p. 361). 

I 318. Clearing Mixture (£281, 298). — (A). One of the most satisfactory and 
generally applicable clearers is carbol turpentine, made by mixing carbolic acid 
crystals (Acidum carbolicnm. A.phenicum crystallizatum) 40 cc. with rectified 
oil of turpentine {Oleum terebinthinae rectification) 60 cc. If the carbolic acid 
does not dissolve in the turpentine add 5 cc. of 95% alcohol, or increase the tur- 
pentine, thus : carbolic acid 30 cc. , turpentine 70 cc. 

(B). Carbol-Xylene Clearer. — Vasale recommends as a clearer, xylene 75 cc, 
carbolic acid (melted crystals) 25 cc. It is used in the same way as the preceding. 

\ 319. Collodion. — This is a solution of soluble cottonf or other form of pyroxy- 
lin in equal parts of sulphuric ether and 95% alcohol. Two solutions are used : 



*The hydrocarbon xylene (C 8 H 10 ) is called xylol in German. In English 
members of the hydrocarbon series have the termination "ene" while members of 
the alcohol series terminate in "ol." 

|The substance used in preparing collodion goes by various names, soluble cot- 
ton or collodion cotton is perhaps best. This is cellulose nitrate, and consists of a 
mixture of cellulose tetranitrate C 12 H r6 (N0 3 ) 4 6 , and cellulose pentanitrate, 
C 12 H 15 (N0 3 ) 5 05. Besides the names soluble and collodion cotton, it is called gun 
cotton and pyroxylin. Pyroxylin is the more general term and includes several 
of the cellulose nitrates. Celloidin is a patent preparation of pyroxylin, more ex- 
pensive than soluble cotton, but in no way superior to it for imbedding. 

Soluble cotton should be kept in the dark to avoid decomposition. After it is 
in solution this decomposition is not so liable to occur. The decomposition of the 
dry cotton gives rise to nitrous acid, and hence it is best to keep it in a box loosely 
covered so that the nitrous acid may escape. 

Cellulose nitrate is explosive under concussion and when heated to 150 centi- 
grade. In the air, the loose soluble cotton burns without explosion. It is said not 
to injure the hand if held upon it during ignition and that it does not fire gun- 
powder if burned upon it. So far as known to the writer, no accident has ever 
occurred from the use of soluble cotton for microscopical purposes. I wish to ex- 
press my thanks to Professor W. R. Orndorff, organic chemist in Cornell Univer- 
sity, for the above information. Proc. Amer. Micr. Soc, vol. XVII (1895), pp. 
361-370. 



CH. VII] PREPARATION OF REAGENTS 201 

(A). 6% to 8% or thick collodion. It is made by mixing 50 cc. of sulphuric 
ether and 50 cc. of 95% alcohol and adding 6 or 8 grams of soluble cotton. If this 
is shaken repeatedly the solution will be complete in a day or two. 

(B). i}4% or thin collodion. To prepare this 1% grams of soluble cotton are 
added to 100 cc. of ether-alcohol (§ 322). 

I 320. %% collodion or cementing collodion. To prepare it ^(ths of a gram 
of soluble cotton is added to alcohol, 60 cc, ether, 40 cc. 

The excess of alcohol prevents the mixture from softening the paraffin (§ 290). 

I 321. Eosin. — This is used mostly as a contrast stain with hematoxylin, which 
is an almost purely nuclear stain. It serves to stain the cell-body, ground sub- 
stance, etc., which would be too transparent and invisible with hematoxylin alone. 
If eosin is used alone it gives a decided color to the tissue and thus aids in its 
stud}* {I 144). Eosin is used in alcoholic and in aqueous solutions. A very satis- 
factory stain is made as follows : 50 cc. of water and 50 cc. of 95% alcohol are 
mixed and i-ioth of a gram of dry eosin added. 

The eosin is used after the hematoxylin in most cases (§ 280), and, as it is in 
alcoholic solution, it may be washed off with 95% alcohol if the object is to be 
mounted in balsam. If it is to be mounted in glycerin or glycerin jelly, the excess 
of eosin should be washed away with distilled water. 

\ 322. Ether, Ether- Alcohol.— Sulphuric ether is meant when ether is men- 
tioned in this book. For the ether-alcohol mentioned in \ 269, 319, etc., a mixture 
of equal volumes of sulphuric ether and 95% alcohol is meant. 

\ 323. Farrants' Solution. — Take 25 grams of clean, dry, gum arabic ; 25 cc. 
of a saturated aqueous solution of arsenious acid ; 25 cc. of glycerin. The gum 
arabic is soaked for several days in the arsenic water, then the glycerin is added 
and carefully mixed with the dissolved or softened gum arabic. 

This medium retains air bubbles with great tenacity. It is much easier to 
avoid than to get rid of them in mounting. 

\ 324. Formaldehyde Dissociator. — This is composed of 2 cc. of a 40% solu- 
tion of formaldehyde in 1000 cc. of water, to which 6 grams of common table salt 
(sodium chlorid) have been added. Formaldehyde as bought in the market is a 
40% solution in water, and is called formol, formalin, formalose and formal, the 
last name being the preferable one. For its use in isolating cells see \ 260. (Micr. 
Bulletin and Sci. News, vol. XII. (1895), pp. 4-5). 

\ 325. Glycerin. — (A). One should procure pure glycerin for a mounting 
medium. It needs no preparation, except in some cases it should be filtered 
through filter paper or absorbent cotton to remove dust, etc. 

For preparing objects for final mounting, glycerin 50 cc. , water 50 cc. , forms 
a good mixture. For many purposes the final mounting in glycerin is made in an 
acid medium, viz., Glycerin 99 cc, Glacial acetic or formic acid, 1 cc. 

By extreme care in mounting and by occasionally adding a fresh coat to the 
sealing of the cover-glass, glycerin preparations last a long time. They are liable 
to be disappointing, however. In mounting in glycerin care should be taken 
to avoid air-bubbles, as they are difficult to get rid of. A specimen need not be 
discarded, however, unless the aif-bubbles are large and numerous. 

\ 326. Glycerin Jelly. — Soak 25 grams of the best dry gelatin in cold water in 
a small agate-ware dish. Allow the water to remain until the gelatin is softened. 
It usually takes about half an hour. When the gelatin is softened, as may be 



202 PREPARATION OF REAGENTS \CH. VII 

readily determined by taking a little in the fingers, pour off the superfluous water 
and drain well to get rid of all the water that has not been imbibed by the gelatin. 
Warm the softened gelatin over a water bath and it will melt in the water it has 
absorbed. Add to the melted gelatin about 5 cc. of egg albumen, white of egg ; 
stir it well and then heat the gelatin in the water bath for about half an hour. Do 
not heat above 75 or So° C, for if the gelatin is heated too hot it will be trans- 
formed into meta-gelatin and will not set when cold. The heat will coagulate the 
albumen and form a kind of floculent precipitate which seems to gather all fine 
particles of dust, etc., leaving the gelatin perfectly clear. After the gelatin is 
clarified it should be filtered through a hot flannel filter and mixed with an equal 
volume of glycerin and 5 grams of chloral hydrate and shaken thoroughly. If it 
is allowed to remain in 'a warm place {i. e,, in a place where the gelatin remains 
melted ) the air-bubbles will rise and disappear. 

In case the glycerin jelly remains fluid or semi-fluid at the ordinary temper- 
ature (i8°-2o° C), the gelatin has either been transformed into meta-gelatin by too 
high a temperature or it contains too much water. The amount of water may be 
lessened by heating at a moderate temperature over a waterbath in an open vessel. 
This is a very excellent mounting medium. Air-bubbles should be avoided in 
mounting as they do not disappear. 

§ 327. Chloral Hematoxylin. — -Hematoxylin is one of the most useful stains 
employed in histology. An excellent solution for ordinary section staining 
may be made as follows : Distilled water 200 cc. , and potash alum 7! grams, are 
boiled together for 5 minutes, in an agate-ware or glass vessel, and sufficient boiled 
water added to bring the water back to 200 cc. After the mixture is cool, 4 grams 
of chloral hydrate, and T 2 oths grams of hematoxylin crystals, previously dissolved 
in 20 cc. of 95% alcohol, are added. The boiling seems to destroy any fungi pres- 
ent in the alum or water, and the chloral prevents the development of any that 
may get in afterward, and this solution therefore is quite permanent. 

At first the color will be rather faint, but after a week or two it will become a 
a deep purple. The deepening of the color is more rapid if the bottle is left un- 
corked in the light and is shaken occasionally. It may be prepared for work 
at once by the addition of a small amount of hydrogen dioxid (H 2 2 ). 

If the stain is too concentrated it may be diluted with freshly distilled water 
or with a mixture of water, alum and chloral. If the stain is not sufficiently con- 
centrated, more hematoxylin may be added. With hematoxylin of the strength 
given in the formula, sections are usually sufficiently stained in from one to five 
minutes. 

As may be inferred from what was said above, the boiling is to destroy any 
living ferments present in the water or alum, and the chloral hydrate is to prevent 
the development of germs which accidentally reach the solution after it is made. 

No precaution is necessary in using this stain for sections, except that appli- 
cable to all hematoxylin solutions, viz : It must be filtered occasionally and after 
staining the surplus stain must be very thoroughly washed away with water ; 
otherwise black granules or needles will appear in or upon the sections. If granules 
appear in the preparations in spite of the washing, it will be well to boil the solu- 
tion three to five minutes and filter through paper or absorbent cotton. The addi- 
tion of one or two per cent, of chloral after the boiling is also advantageous. 
This stain has not been tried for dyeing in bulk. Other substances than chloral 



CH. VII] PREPARATION OF REAGENTS 203 

were tried, but not with so good success. (Proc. Amer. Micr. Soc, 1892, pp. 
125-127). 

\ 328. Hematein. This is used instead of hematoxylin, as it is believed to 
give more satisfactory results. Prepare as follows : Prepare a 5% solution of pot- 
ash alum in distilled water and boil or leave in a steam steralizer an hour or two. 
While this alum solution is warm add 1 per cent, of hematein dissolved in a small 
quantity of alcohol. After the fluid has cooled add 2 grams of chloral for each 
100 cc. of solution. (Freeborn, Jour. Ap. Micr., 1900, p. 1056.) 

\ 329. Liquid Gelatin. — Gelatin or clear glue, 75 to 100 grams. Commercial 
acetic acid (No. 8) 100 cc. , water 100 cc, or glacial acetic acid 40 cc. and water 
160 cc, 95% alcohol 100 cc, glycerin 15 to 30 cc. Crush the glue and put it into 
a bottle with the acid, and set in a warm place, and shake occasionally. After 
three or more days add the other ingredients. This solution is excellent for 
fastening paper to glass, wood or paper. The brush must be mounted in a quill 
or wooden handle. For labels, it is best to use linen paper of moderate thickness. 
This should be coated with the liquid gelatin and allowed to dry. The labels may 
be cut of an3^ desired size and attached by simply moistening them, as in using 
postage stamps. 

Very excellent blank labels are now furnished by dealers in microscopical 
supplies, so that it is unnecessary to prepare them one's self, except for special 
purposes Those like that shown in Fig. 163 may be had for about $4 for 10,000. 

\ 330- Nitric Acid Dissociator. — This is prepared by mixing So cc. of water 
with 20 cc. of strong nitric acid. It is used mostly in dissolving the connective 
tissue of muscle and thus making it possible to separate the fibers. Alum water 
is used as a restrainer (2 314 and 264). (Proc. Amer. Micr. Soc, Vol. XI, ( 1889), 
pp. 34-45)- 

\ 33 r - Normal Salt Solution or Saline Solution. — Pure water from its differ- 
ing density from the natural lymph acts injuriously on the tissues. The addition 
of a little table salt, however, prevents this deleterious action, or greatly lessens it, 
hence the name of normal salt solution. It is a T %% solution of table salt (sodium 
chlorid) in water ; water 1000 cc. , salt 6 grams, or water 100 cc, salt j\ gram. 

\ 332. Paraffin. Paraffin is of various melting points, hence at the ordinary 
temperature of a laboratory, that melting at the lowest temperature will be moder- 
ately soft, hence soft paraffin, while that melting at a higher temperature will be 
hard. For the best results one usually has to mix hard and soft paraffins. The 
larger the object to be cut and the thicker the sections the softer should be the 
paraffin. 

\ 333. Picric-Alcohol. — This is an excellent hardener and fixer for almost all 
tissues and organs. It is composed of 500 cc of water and 500 cc. of 95%' alcohol, 
to which 2 grams of picric acid have been added. (It is a \% solution of picric 
acid in 50% alcohol). It acts quickly, in from one to three days. (§ 267, 2S4). 
(Proc. Amer. Micr. Soc, Vol. XII (1890), pp. 120-122). 

\ 334. Shellac Cement. — Shellac cement for sealing preparations and for mak- 
ing shallow cells f| 248) is prepared by adding scale or bleached shellac to 95% 
alcohol. The bottle should be filled about half full of the solid shellac then enough 
95% alcohol added to fill the bottle nearly full. The bottle is shaken occasionally 
and then allowed to stand until a clear stratum of liquid appears on the top. This 
clear, supernatant liquid is then filtered through absorbent cotton, using a paper 



204 PREPARATION OF REAGENTS \CH. VII 

funnel (§ 315), into an open dish or a wide-mouth bottle. To every 100 cc. of 
this filtered shellac, 2 cc. of castor oil and 2 cc. of Venetian turpentine are added 
to render the shellac less brittle. The filtered shellac will be too thin, and must 
be allowed to evaporate till it is of the consistency of thin syrup. It is then put 
into a capped bottle, and for use, into a small spirit lamp (Fig, 153). In case the 
cement gets too thick add a small amount of 95% alcohol or some thin shellac. 
The solution of shellac almost always remains muddy, and in most cases it takes a 
very long time for the flocculent substance to settle. One can very quickly obtain a 
clear solution as follows : When the shellac has had time to thoroughly dissolve, 
i. f., in a week or two in a warm place, or in less time if the bottle is frequently 
shaken, a part of the dissolved shellac is poured into a bottle and about one-fourth 
as much gasolin or benzin added and the two well shaken. After twenty-four 
hours or so the flocculent, undissolved substance will separate from the shellac so- 
lution and rise with the gasolin to the top. The clear solution may then be siphoned 
off or drawn off from the bottom if one has an aspirating bottle. (R. Hitchcock, 
Amer. Monthly Micr. Jour , July, 1884, p. 131). 

If one desires to color the shellac, the addition of a strong alcoholic solution 
of some of the coal tar colors is good, but it is liable to dissolve in the mounting 
medium when shellac is used for sealing. A small amount of lampblack well 
rubbed up in very thin shellac and filtered, is good to darken the shellac. 

ARRANGING AND MOUNTING MINUTE OBJECTS 

\ 335. Minute objects like diatoms or the scales of insects may be arranged in 
geometrical figures or in some fanciful way, either for ornament or more satisfac- 
tory study. To do this the cover-glass is placed over the guide. This guide for 
geometrical figures may be a net-micrometer or a series of concentric circles. In 
order that the objects may remain in place, however, they must be fastened to the 
cover-glass. As an adhesive substance, mucilage or liquid gelatin (£ 329) thinned 
with an equal volume of 50% acetic acid answers well. A very thin coating of this is 
spread on the cover with a needle, or in some other way and allowed to dry. The ob- 
jects are then placed on the gelatinized side of the cover and carefully got into posi- 
tion with a mechanical finger, made by fastening a cat's whisker in a needle holder. 
For most of these objects a simple microscope with stand (Figs. 20, 145, 146) will be 
found of great advantage. After the objects are arranged, one breathes very gently 
on the cover-glass to soften the mucilage or gelatin. It is then allowed to dry and 
if a suitable amount of gelatin has been used, and it has been properly moistened, 
the objects will be found firmly anchored. In mounting one may use Canada bal- 
sam or mount dry on a cell {\ 247, 255). See Newcomer, Amer. Micr. Soc.'s Proc, 
1886, p. 128 ; see also E. H. Griffith and H. L. Smith, Amer. Jour, of Micros., iv, 
102, v, S7 ; Amer. Monthly Micr. Jour., i, 66. 107, 113. Cunningham, The Micro- 
scope, viii, 1888, p. 237. 



For additional apparatus for this chapter, see Ch. X. 



CHAPTER VIII 



PHOTOGRAPHING OBJECTS WITH A VERTICAL CAMERA ; 
PHOTOGRAPHING LARGE TRANSPARENT OBJECTS ; 
PHOTOGRAPHING WITH A MICROSCOPE : (A) TRANS- 
PARENT OBJECTS ; (B) OPAQUE OBJECTS AND THE 
SURFACES OF METALS AND ALLOYS* 



APPARATUS AND MATERIAL FOR THIS CHAPTER 

Vertical camera with photographic objectives (Fig. 169), small vertical camera 
with special microscope stand for embryos, etc. (Figs. 183-184); arrangement of 
camera for large transparent objects (Fig. 181); photo-micrographic cameras (Figs. 
183-1S4, 192); photographic objectives for gross and microscopic work (Figs. 170- 
171, 176-1S0); microscope, microscopic objectives and projection oculars (Figs. 185, 
189, 193); color screens, lamps, dry plates and the chemicals and apparatus neces- 
sary for developing, printing, etc. 

\ 336. Nothing would seem more natural than that the camera, armed with a 
photographic objective or with a microscopic objective, should be called into the 
service of science to delineate with all their complexity of detail, the myriads of 
forms studied. 

For photographing many objects in nature the camera remains horizontal or 
approximately so, but for a great many of those studied in botany, zoology, miner- 
alogy and in anatomy the specimens cannot be put in a vertical position necessary 
for a horizontal camera. This difficulty has been overcome by using a mirror or a 
45-degree prism. These are practically alike and have the defect of producing a 
picture with the inversion of a plane mirror. 

VERTICAL CAMERA 

\ 2,2,7- To meet all the difficulties the object may be left in a horizontal posi- 
tion and the camera made vertical (fig. 169). 

For the last twenty-five years such a camera has been in use in the Anatomical 
Department of Cornell University for photographing all kinds of specimens ; 



* Papers on this subject were given by the writer at the meeting of the Amer- 
ican Association for the Advancement of Science in 1879, and at the meeting of 
the Society of Naturalists of the eastern United States in 1883 ; and in Science 
Vol. Ill, pp. 443, 444. 



206 PHOTO-MICROGRAPHY [CH. VIII 

among these, fresh brains, and hardened brains have been photographed without 
the slightest injury to them. Furthermore, as many specimens are so delicate 
that they will not support their own weight, they may be photographed under 
alcohol or water with a vertical camera and the result will be satisfactory as a 
photograph and harmless to the specimen. 

A great field is also open for obtaining life-like portraits of water animals. 
Freshly killed or etherized animals are put into a vessel of water with a contrast- 
ing back ground and arranged as desired, then photographed. The fins have 
something of their natural appearance and the gills of branchiate salamanders 
float out in the water in a natural way. In case the fish te*nds to float in the water 
a little mercury injected into the abdomen or intestine will serve as ballast. 

The photographs obtainable in water are almost if not quite as sharp as those 
made in air. Even the corrugations on the scales of such fishes as the sucker 
(Catostomus teres) show with great clearness. Indeed so good are the results 
that excellent half tone plates may be produced from the pictures thus made, also 
excellent photogravures. In those cases, as in anatomical preparations, where the 
photograph rarely answers the reqivirements of a scientific figure, still a photo- 
graph serves as a most admirable basis for such a figure. The photograph is made 
of the desired size and all the parts are in correct proportion and in the correct 
relative position. From this photographic picture may be traced all the outlines 
upon the drawing paper, and the artist can devote his whole time and energy to 
giving the proper expression without the tedious labor of making measurements. 

"While the use of photography for outlines as bases for figures diminishes the 
labor of artists about one-half it increases that of the preparator ; and herein lies 
one of its chief merits. The photographs being exact images of the preparations, 
the tendency will be to make them with greater care and delicacy, and the result 
will be less imagination and more reality in published scientific figures ; and the 
objects prepared with such care will be preserved for future reference." 

"In the use of photography for figures several considerations arise : (i) The 
avoidance of distortion ; (2) The adjustment of the camera to obtain an image of 
the desired size ; (3) Focusing ; (4) Lighting and centering the object. 

(1). While the camera delineates rapidly, the image is liable to distortion. 
I believe opticians are agreed, that, in order to obtain correct photographic images, 
the objective must be properly made, and the plane of the object must be parallel 
to the plane of the ground glass. Furthermore, as most of the objects in natural 
history have not plane surfaces, but are situated in several planes at different 
levels, the whole object may be made distinct by using in the objective a dia- 
phragm with a small opening. 

§ 33S. Scale of Sizes and Focusing. — (2). By placing the camera on a long 
table, and a scale of some kind against the wall, the exact position of the ground 
glass for various sizes may be determined once for all, and these positions 
noted in some way. 

Fig. 169. Vertical Camera for photographing objects in a horizontal position. 
The camera is attached to a double frame connected by bent metal pieces 
fastened to the lower and sliding in a groove hi the upper frame. The two frames 
can then slide over each other without separating . For moving the outer frame a 
rack work is put on the lower or inner frame and a pinion with a toothed wheel on 
the outer one. This is turned by the wheel shown. To prevent the camera run- 



CH. VIII '] 



PHO TO-MICR OGRAPHY 



207 



ning down in the vertical position a pawl is held in place by a spring. This may 
be released by a smaller wheel than that serving to move the pinion. This rack and 
pinion are fine enough for focusing with the photographic objectives employed. 




Fig. 169. 

The camera bed is graduated in centimeters so that the exact extent of the 
bellows can be determined by inspection. 

The support on which the specime?i rests is of heavy glass on vertical 
rods about 10 centimers long. The background is placed on the table top about 10 
cm. below. This arrangement of support and background serves to avoid the dense 
shadows which make it difficult to determine exactly the limits of the specimen. 
To make the apparatus steady the right hand end of the table is heavily weighted. 
The tables have leveling screws in the legs. 



208 



PHO TO-MICROGRAPHY 



[CH. VIII 




Fig. 170. Turner-Reich 
anastigmat objective for pho- 
tography, ( Gundlach Opt. 

Co.) 

In the camera here fig- 
ured, the camera bed is ruled 
in centimeters so that the 
position of the ground glass 
can be determined with ac- 
curacy and noted. It takes 
but a moment to set the 
ground glass or focusing 
screen at the right level to 
give any desired size. In 
practice it is convenient to 
have attached to the camera 
a table giving the position of 
the ground glass for various 
sizes, and also the distance of 
the objective from the object 
in each case. By having this 
information it takes but a 
moment to set the camera 
and to place it so that it 
will be approximately i n 



Fig. 170. 



Fig. 171. Zeiss anastigmat objective 
for photography, (Bausch & Lomb Opti- 
cal Co. ) 

focus. The final focusing is then accom- 
plished by the use of the rack and pinion 
movement. It is an advantage to use a 
focusing glass and a clear focusing screen 
or the transparent part of the ordinary 
screen (Fig. 174), for the final focusing. 
As many objects have not sharp details Fig. 171. 

which one can focus on, it is helpful to 

place some printed letters on the part to be brought out with the greatest sharp- 
ness. Of course these are removed before the exposure is made. 

\ 339. In lighting the object one should take pains to so arrange it with ref- 
erence to the light that the details will show with the greatest clearness. Naturally 
for the vertical camera the light will come from the side and not from a skylight, 
although good results are obtained with a skylight if one so places the camera that 
it does not cast objectionable shadows. 




CH. VIII} 



PHO TO-MICROGRA PH Y 



209 




Fig. 172. 



Fig. 172. Tripod magnifier as focusing glass. This 
is carefully focused on a scratch or pencil mark on the 
lower or ground surface of the focusing screen. Then 
whenever the object is sharply focused the focal plane will 
be at the level of sensitive surface. 

As shown in Fig. 169, the object is placed upon a 
glass support and the background is quite a distance 
below the support. For a dark object the background 
should be light, and for a light one dark. Black 
velveteen is excellent for a back-ground. The advan- 
tage of the glass support is that the shadows in the 
background which often make it difficult to tell just where the specimen ends and 
the background begins, is wholly done away with, and that too without at all 
affecting the proper light and shade of the object itself. (Method of W. E. 
Rumsey, Canadian Entomologist 1896, p. S4). 



Fig. 173. Focusing Glass. "It is achromatic, 
consisting of a double convex crown lens and a nega- 
tive meniscus flint lens cemented together.''' It screws 
into the brass tube and is thus adjustable, enabling one 
to focus the pe?icil mark in the clear area of the focus- 
ing screen (Fig. ijf) zvith great accuracy. It also 
serves to focus the image with ease and accuracy. The 
eye must not be too close to the tipper end of the focus- 
ing glass or the field will be restricted. (Gundlach 
Opt. Co.) 



Fig. 173. 

Fig. 174. Ground-glass focusing screen 
with central transparent area for exact 
focusing with a focusing glass when one 
does not possess a clear focusing screen. (/) 
The ground surface ; (2) Central part with 
oblong cover-glass and Canada balsam on the 
ground surface to render it transparent. X. 
The central point in the entire focusing 
screen. It is made with a black lead pencil 
on the grotmd surface. The focusing glass 
is focused on this cross, then when the image 
is in focus it will be at the level of the sen- 
sitive coating of the plate. 






1 






2 

X 






Fig. 175. 



CH. VIII] PHOTO-MICROGRAPHY 21 1 

Fig. 175. Vertical Camera and special microscope stand for photographing 
embryos and other small specimens in liquids and for photographing large sections. 
The camera rests on a low table and the operator can stand on the floor while per- 
forming all the operations. 

The stage of the microscope is attached to the arm in the place of the tube. 
This stage has two stories. The specimen is shown on the upper and the back- 
ground on the lower story. 

In focusing, the coarse and fine adjustment of the special microscope stand are 
used. The large mirror is to illuminate embryo chicks mounted entire, and other 
large transparent preparations. Trans. Amer. Micr. 1901. 

\ 340. Prints. — If the photographic prints are to be used solely for outlines, 
the well-known blue prints so much used in engineering and architecture may be 
made. If, however, light and shade and fine details are to be brought out with 
great distinctness, either an aristotype, platinotype or a bromide print is preferable. 

§ 341. Recording, Storing and Labeling Negatives. — In or- 
der to get the greatest benefit from past experience it is necessary to 
make the results available by means of a careful record. For this pur- 
pose the table (§ 360) has been prepared. If one gives the information 
called for in this table, whether the result is successful or not, one can 
after a time work with great exactness, for the elements of success and 
failure will stand out clearly in the table. 

§ 342. Labeling the Negatives. — After the negative is dry the 
labeling can be done on the gelatin side with carbon ink. Enough 
data should be given to enable the certain identification of the negative 
at any future time. 

§ 343. Storing Negatives. — This is satisfactorily done by put- 
ting each into an envelope and writing a duplicate label on the upper 
edge, and then the negatives may be placed in drawers in alphabetical 
order as are the catalog cards of books in a library. One can then find 
any negative with the same facility that the title of a book can be 
found in a card catalog. 

PHOTOGRAPHING EMBRYOS 

For photographing embryos and many other small specimens it is 
more convenient to use a smaller apparatus than the vertical camera 
just described. It is necessary also to have a more delicate method of 
focusing. 

§ 344. Camera for Embryos. — This is a vertical camera for 
photographing with the microscope, with the photographic objective in 
the end of the camera as for an ordinary camera. This is readily ac- 
complished by having a society screw adapter, and also adapters for 
the micro-planars or other objectives which one desires to use. The 



212 



PHO TO-MICROGRAPHY 



[ CH. VIII 




magnification usually required varies from natural size (X i) to five 
times natural size ( X 5) up to X 20. As with the large camera the 
position of the ground glass for each magnification and for each objec- 
tive is determined once for all by using a scale in millimeters. The 
various positions are accurately noted, then one can set the camera 
almost instantly for the desired magnification. The supporting rod is 
divided to half centimeters and therefore the exact position is easily 
recorded (Fig. 183). 

Fig. 176. Zeiss Micro-Planar for photographing 
with low magnification and for projection (see Ch. IX). 
These are made from 20 to 100 mm. equivalent focus, 
those of 20 and 35 mm. equivalent focus have the standard, 
Royal Society Screw, the others have a larger screw in- 
order that the image may not be restricted. ( Cut loaned 
by Bausch & Lomb Optical. Co.) 

§ 345. Special Microscope Stand. — For the accurate focusing 
necessary for embryos one should possess a special microscope 
stand with the stage in two or three stories and attached to the arm in 
place of the tube of the microscope. The stage proper is absent. 
This arrangement of the stage permits the use of the coarse and fine 
adjustment of the microscope to be used for focusing. The position of 
the camera on a low table (45 to 50 cm. high; makes it possible for the 
operator to stand on the floor while making all the adjustments of the 
the embryo and for focusing ; and all the parts are within reach 
(Fig. 175). 

§ 346. Arranging the Embryos.— As usually prepared the em- 
bryos are white and therefore require a dark background. This may 
be attained either by placing the embryos in a dark dish or on some 
paper blackened with water-proof India ink, or by/ putting them in a 
glass vessel like a Petri dish, and a piece of black velveteen on the 
stage below\ The specimens will of course be in a liquid, usually alcohol. 




Fig. 177. Wide angle anastigmat 
objective for photographing at low mag- 
nification (Bausch & Lomb Optical Co.). 



If several embryos are to be 
taken at once, the embryos are ar- 
ranged in rows something as the 
words on a line. Arrange them 
in even vertical as well as horizontal rows so that when the print 
is made it will be easy to cut them apart. When the embryos are ar- 



CH. VIII '] PHOTO-MICROGRAPHY 213 

ranged, one should be certain that the light brings out the details most 
desired. For example, if one is photographing an embryo which 
shows the branchial pockets well, great pains should be taken to so 
arrange the embryo with reference to the light that the proper shading 
will be given to bring out the gill pockets most emphatically. One can 
learn to do this only by practice. It is advantageous to have an assist- 
ant, then while the operator is looking into the camera the assistant 
can turn the embryo in various directions until the appearance is most 
satisfactory. 

§ 347. Focusing and Making the Exposure. — For getting a 
general focus, and for the general arrangement the ground glass screen 
is used, but for the final focusing it is desirable to use a clear glass 
screen and a focusing glass. In this way one can focus as satisfactor- 
ily as with an ordinary microscope. In daylight with white embryos 
and a dark ground 30 to 40 seconds is usually sufficient exposure. One 
must learn this also by trial and it facilitates the obtaining of exact data 
to make a record of every negative made, whether the negative is good 
or bad. A table is given in § 360 to facilitate the record taking. In a 
short time one can learn to make the correct exposure. If the result 
is unsatisfactory, try again. The rule adhered to by all first rate 
workers is to to stick to it until the result is satisfactory. 

§ 348. Records of Embryos. — Each specimen or litter of speci- 
mens will have its own label giving date and method of preparation. 
It is an advantage to write this label with water-proof carbon ink, then 
one can put the label in the dish with the embryos and it will form a 
part of the picture and serve as a record. 

After the picture is satisfactorily made it is wise to number the 
embryos on the back of the negative with a wax crayon, and later 
when the negative is dry number on the front with carbon ink. The 
embryos are placed in separate bottles each with a copy of the original 
label and the number corresponding with that put on the negative. 
This is easily accomplished if the embryos are arranged in definite 
rows as advised in § 346. 

Finally when the embryo is cut into serial sections and mounted, 
a picture of the whole embryo should accompany the series. 

§ 349. Size of the Pictures. — For all embryos it is well to 
make one picture natural size (x 1) and then for the smallest ones a 
magnification of at least five times natural size (x 5). Here, as with 
the magnification of the microscope, linear magnification is always 
meant (§ 154-155). 



214 



PHO TO-MICROGRAPH Y 



[CH. VIII 



§ 350. — Objectives. — For making pictures from one to five times 
natural size objectives of 60 to 100 mm. focus answer well (Figs. 1 76— 
180). Short focus (75 to 100 mm. equivalent focus), wide angle pho- 
tographic objectives are also admirable for this work. 




V 

42. 

E.LE 
WET 

1 


~y. 


f""^ "l 


nil' 1 ; 

!TI, 
LAR 






Fig. 178. Fig. 179. 
Fig. 178. Leitz 64 millimeter ob- Fig. 179. Leitz 42 millimeter ob- 
jective for photography and for projec- jective for photography and for projection 
tion ( Wm, Krafft, N. Y.) ( Wm. Krafft, N. Y.) 

P'iG. 180. Zeiss' Apochromatic Projection Objective of 70 
mm. equivalent focus, for photo-micrography. (Zeiss' Catalog.) 
This, and another of 35 mm. focus, are designed for making 
8 pictures of moderate magnification. Usually rather large ob- 
jects -are photographed with them. The object may be illumina- 
ted in the ordinary way. They are used without an ocular, 
like a photographic objective. The one of 35 mm. is screwed 
into the tube of the microscope like an ordinary objective, but the 
one of 70 mm. here shown, is, by means of a conical adapter, 
screwed into the ocular end of the tube, Fig. iSg. 

For illuminating the object, any suitable light may be used, 

but it is recommended that the light be concentrated by means 

of a bull's eye or some form of combination like the engraving 

glass, and that the condenser be so placed that it focuses the light tipon the objective, 

not upon the object. The object is then illuminated with a converging cone of light. 

§ 351. Record of Negatives. — As indicated in § 341-343 each 
negative should have a record, see record blank on p. 219. On the 
negative itself should be also written the main facts with carbon ink. 
The name and magnification, date and any other details which may be 
thought desirable can be put on the envelope containing the negative, 
and then the negative stored like a catalog card as described above 
(§ 343)- 

PHOTOGRAPHING LARGE TRANSPARENT OBJECTS 

§ 352. There are many large transparent objects which it is de- 
sirable to photograph, e. g., chick embryos mounted whole, large sec- 
tions of organs like the brain, etc. These must be photographed at a 
low magnification. 



CH. VIII} 



PHO TO-MICROGRAPHY 



215 




Fig. i Si. 

Fig. 181. Camera and special microscope stand for photographing very large 
transparent sections. For this the vertical camera is used {Fig. 169) with the 
camera reversed on the sliding frame. This frame is elevated sufficiently to utilize 
the sky as background and illuminant. The special microscope stand is inclined to 
the horizontal and placed on the fixed frame supporting the camera ; the specimen 



216 PHOTO-MICROGRAPHY [CH VIII 

placed on the stage. For objective one of the objectives shown in Figs. ij6 to /So is 
used. The objective is screwed into an adapter in place of the ordinary photographic 
objective. The focusing is performed roughly by the rack and pinion, and then 
with great exactness with the focusing glass. For manipulating the fine adjust- 
ment of the special microscope the well known device of a cord over the head of the 
micrometer screw is used. (See also Fig. 175.) ( Trans. Amer. Micr: Soc, 1001.) 

Successful photographs require an even lighting and an objective 
which has sufficient field to take in the whole object. The camera 
used for embryos (Fig. 175) answers very well for objects of moderate 
size. For lighting them the specimen is put on the upper stage, and the 
back-ground shown in the figure is removed. Then the large mirror 
is used to throw light up through the preparation. If necessary the 
specimen can be placed on one of the lower stories to bring it nearer 
the mirror. The lighting and focusing should be as perfect as possi- 
ble. Lamplight and daylight are both good. 

§ 353- Photographing Large Transparent Objects. — For this 
the large vertical camera (Fig. 169, 181) is reversed in position on the 
supporting frames, and elevated only sufficiently to make a sky back- 
ground ; or a 45 degree reflector of white cloth or paper of sufficient 
size must be used for a horizontal camera. If one has the earth for 
back-ground the light will be dull and uneven and a very long expos- 
ure is necessary, and the final results unsatisfactory. 

§ 354. Use of the Special Microscope Stand. — In order to 
hold the specimen in position and to focus it accurately, it is put on 
the stage of the special microscope stand (Fig. 175), which is inclined, 
and fastened to the fixed part of the frame supporting the camera. As 
the stage of this microscope is moved by the coarse or the fine adjust- 
ment, the focusing can be accomplished with the same accuracy as the 
microscope itself. For the general arrangement of the specimen and 
the rough focusing the ground glass is used, then this is replaced by a 
clear-glass focusing screen, and by the aid of a focusing glass the speci- 
men is put in perfect focus. As one cannot reach the fine adjustment 
while focusing, the well known device of a cord over the head of the 
micrometer screw is resorted to. The two ends of the cord should be 
weighted with about 50 or a hundred grams to keep the cord taut, then 
whichever one is pulled, the micrometer screw will respond at once. 
To cut off the light a piece of black velveteen is hung over the end of 
the objective. This can be removed without jarring the apparatus. 
An exposure of a few seconds (3 to 10 seconds), will suffice for many 
preparations, unless a color screen is used. The color screen increases 
the time of exposure from three to five times (§ 359). 



CH. VIII] 



PHO TO-MICROGRAPHY 



217 




Fig. 182. Bulls eye lens and holder. (Bausch & Lomb Opt. Co. ) 



COLOR-CORRECT PHOTOGRAPHY 



From the fact that the different wave lengths of light affect the photographic 
plate with different degrees of vigor, the ordinary photographic print of a many 
colored object or landscape is not satisfactory. All objects whose light is of short 
wave lengths, as blue, etc., will appear too light and those which are red, yellow 
and green will be too dark relatively. To obviate this difficulty two methods have 
been adopted, and for the most complete success they must be combined. 

(A) The use of ortho- or iso- chromatic plates and (B) the use of a color screen 
or light filter. 

\ 355- Orthochromatic or Isocbromatic Plates. — These are plates which have 
been rendered much more sensitive than ordinary plates to the long waves of red, 
orange, yellow and green, they therefore give a much more natural rendering to 
many-colored objects than ordinary plates. As they are sensitive to red, orange, 
etc. , one must be very careful in exposing them in the dark room even to the light of 
the developing lantern. The more nearly the plate can be kept from all light, 
except that acting during the exposure in the camera, the more satisfactory will 
be the resulting negative. 

These color-correct plates are not very enduring, and must be used while they 
are fresh, or only weak, foggy negatives will result. 



218 PHOTO-MICROGRAPHY [CH. VIII 

For photographing transparent or translucent objects there is a further diffi- 
culty introduced, viz, greater or less transparency. Assuming that all the rays of 
the spectrum are equally active, there would still be difficulty because the blue 
and violet stains used in microscopy are liable to be more transparent than those 
stained with red and orange, consequently a blue stained preparation is liable to 
lack in contrast since the light reaching the plate from the object and from the 
space around it produce so nearly equal effects on the plate. 

On the other hand a red stain gives too much contrast because the light pass- 
ing through it has little effect as compared with the light going through the space 
surrounding the object. So far in the discussion it is assumed that the objects 
are practically transparent and that pure red and blue are used. As a matter of 
fact the stain renders the specimen somewhat more opaque so that the specimen 
would be darker than the background in either case. This is especially true of 
hematoxylin. While hematoxylin is blue or purple, it renders the tissues more or 
less opaque, so that with a petroleum lamp and isochromatic plates, freshly stained 
hematoxylin specimens are very easy to get good pictures of. 

\ 356. Color Screens. — As a general statement all color screens which have 
proved really useful in photographing transparent or translucent microscopic 
specimens cut off most of the blue end of the spectrum. Others cut off also the 
red end, leaving only the middle, visually brightest part of the spectrum free. As 
modern achromatic objectives are corrected for the visual rays, a screen cutting 
off the blue end of the spectrum serves to obviate any lack of sharpness due to 
the aberration of the blue rays in such objectives. There is the further advan- 
tage that with red and yellow objects, the color being in general of the wave 
length transmitted by the screen would be in true relative shade or contrast be- 
tween background and object, and give good detail in the object. For the blue 
object this form of screen is also good for, as it cuts off the blue rays, the effect 
will be like photographing a gray object where the light and shade depend on the 
transparency of different parts. The denser the part the more opaque it is and 
therefore the darker it appears with transmitted light. The background allowing 
all the colored light to pass is lighter than the blue object and therefore there will 
be good contrast and also good detail in the object. 

§ 357 Composition of Color Screens. — The most successful color screens are 
solutions held in parallel sided glass vessels. Colored glass, gelatin, collodion, 
etc., colored with different chemicals are fairly satisfactory, but not so satisfac- 
tory as the solutions. 

(1) The most generally used and most generally useful screen is a watery 
solution of dichromate of potash (K 2 Cr 2 7 ). This cuts off the violet, the blue 
and the bluish green. The amount of light cut out depends upon the density of the 
solution and the thickness of the stratum through which the light passes. 

(2) Zetlnow' s cupro-chromate color screen allows wave lengths from 0.570^ 
to 0.550M to pass, that is yellow light. It is made by dissolving in 250 cc. of water, 
1 1 grams of pure dry cupric nitrate and 1 gram of chromic acid. The thickness of 
the stratum of liquid should be about 1 centimeter. 

(3) Gifford's color screen. This is highly spoken of both for observation 
with the microscope (Carpenter-Dallinger) and for use in photography (J. R. M. 
S. 1S94, p. 164). It is composed of a strong solution of malachit green in water, 
glycerin, glycerin jelly, etc. Only a thin stratum such as could be mounted be- 



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220 PHOTO-MICROGRAPHY [CH VIII 

tween two cover-glasses is needed. By combining a little picric acid with, the 
solution or by the use of a thin piece of signal green glass only light between the 
fixed lines E and F, is allowed to pass. This is not therefore so generally useful 
as dichromate. 

(4) Bothamley" s aurantia color screen is a saturated alcoholic solution of the 
aurantia added to collodion of 3 to 4%. The collodion is poured on a large cover- 
glass or a glass plate and allowed to dry. Pringle advises several screens of aurantia 
of different shades. That is easily managed by adding a greater or less amount of 
the solution to the collodion. This is a good screen and easily used. 

Petroleum light serves as a yellow color screen, and one can often get excel- 
lent results with such a light when daylight or the electric light without a color 
screen does not give a good picture. For all photography with the microscope 
isochromatic or orthochromatic plates are advised. For many objects no color 
screen is needed if one uses a petroleum lamp. 

\ 358. Position of the Screen. — It does not make much difference where the 
color screen is placed provided no light reaches the object which has not passed 
through the screen. 

I 359. Exposure with a Color Screen. — The interposition of a color screen 
increases the time of exposure from three to five times. One can learn the time 
and whether or not to use a color screen, and the kind of a screen to use only by 
experiment. To get the full benefit of these experiments for future work, every 
negative should be carefully recorded (§ 360, table). It would also aid one materi- 
ally, in the beginning at least, if he were to study the color screen used with the 
micro-spectroscope and determine the wave lengths which are allowed to pass 
through it (§ 195, 202). If this study were supplemented by a spectroscopic ex- 
amination of the object to be photographed, one would learn to choose with great 
accuracy the color screen which would give the best results. 

PHOTOGRAPHING WITH A MICROSCOPE* 

§ 361. The first pictures made on white paper and white leather, sensitized 
by silver nitrate, were made by the aid of a solar microscope ( 1802). The pictures 

Considerable confusion exists as to the proper nomenclature of photography 
with the microscope. In German and French the term micro-photography is very 
common, while in English photo-micrography and micro-photography mean dif- 
ferent things. Thus : A photo-micrograph is a photograph of a small or microscopic 
object usually made with a microscope and of sufficient size for observation with 
the unaided eye ; while a micro-photograph is a small or microscopic photograph 
of an object, usually a large object, like a man or woman and is designed to be 
looked at with a microscope. 

Dr. A. C. Mercer, in an article in the Proc. Amer. Micr. Soc, 1886, p. 131, says 
that Mr. George Shadboltmade this distinction. See the Liverpool and Manches- 
ter Photographic Journal (now British Journal of Photography) , Aug. 15, 1858, p. 
203 ; also Sutton's Photographic Notes, Vol. Ill, 1858, pp. 205-20S. On p. 208 of 
the last, Shadbolt's word "Photomicrography" appears. Dr. Mercer puts the 
case very neatly as follows : " A photo-micrograph is a macroscopic photograph of 
a microscopic object ; a micro-photograph is a microscopic photograph of a macro- 
scopic object. See also Medical News, Jan. 27, 1894, p. 108. 



CH. VIII] PHOTO-MICROGRAPHY 221 

were made by Wedgewood and Davy, and Davy says : "I have found that images 
of small objects produced by means of the solar microscope may be copied without 
difficulty on prepared paper. "f 

Thus among the very first of the experiments in photography the microscope 
was called into requisition. And naturally, plants and motionless objects were 
photographed in the beginnings of the art when the time of exposure required 
was very great. 

At the present time photography is used to an almost inconceivable degree in 
all the arts and sciences and in pure art. Even astronomy finds it of the greatest 
assistance. 

It has also accomplished marvels in the production of colored plates for book 
illustrations, especially in natural history. For an example see Comstock's Insect 
Life, 2d edition. 

Although first in the field, Photo-Micrography has been least successful of 
the branches of photography. This is due to several causes. In the first place, 
microscopic objectives have been naturally constructed to give the clearest image 
to the eye, that is the visual image as it is sometimes called, is for microscopic ob- 
servation, of prime importance. The actinic or photographic image, on the other 
hand, is of prime importance for photography. For the majority of microscopic 
objects transmitted light ( \ 64) must be used, not reflected light as in ordinary vis- 
ion. Finally, from the shortness of focus and the smallness of the lenses, the 
proper illumination of the object is accomplished with some difficulty, and the 
fact of the lack of sharpness over the whole field with any but the lower powers, 
have combined to make photo-micrography less successful than ordinary macro- 
photography. So tireless, however, have been the efforts of those who believed in 
the ultimate success of photo-micrography, that now the ordinary achromatic ob- 
jectives with ortho-chromatic or isochromatic plates and a color screen or petrol- 
eum light give good results, while the apochromatic objectives with projection 
oculars give excellent results, even in hands not especially skilled. The problem 
of illumination has also been solved by the construction of achromatic and apoch- 
romatic condensers and by the electric and other powerful lights now available. 
There still remains the difficulty of transmitted light and of so preparing the 
object that structural details stand out with sufficient clearness to make a picture 
which approaches in definiteness the drawing of a skilled artist. 

The writer would advise all who wish to undertake photo-micrography seri- 
ously, to study samples of the best work that has been produced. Among those 
who showed the possibilities of photo-micrographs was Col. Woodward of the U. 



tin a most interesting paper by A. C. Mercer on "The Indebtedness of Pho- 
tography to Microscopy," Photographic Times Almanac, 1887, it is shown that : "To 
briefly recapitulate, photography is apparently somew 7 hat indebted to microscopy 
for the first fleeting pictures of Wedgewood and Davy [1802], the first methods of 
producing permanent paper prints [Reede, 1 837-1 839], the first offering of prints 
for sale, the first plates engraved after photographs for the purpose of book illus- 
tration [Donne &Foucalt, 1845], the photographic use of collodion [Archer & Dia- 
mond, 1851], and finally, wholly indebted for the origin of the gelatino-bromide 
process, greatest achievement of them all [Dr. R. L. Maddox, 1871]. See further 
for the history of Photo-micrography, Neuhauss, also Bousfield. 



222 



PHO TO- Ml CROGRA PH Y 



\CH. VIII 



S. Army Medical Museum. The photo-micrographs made by him and exhibited 
at the Centennial Celebration at Philadelphia in 1876, serve still as models, and uo 
one could do better than to study them and try to equal them in clearness and 
general excellence. According to the writer's observation no photo-micrographs 
of histological objects have ever exceeded those made by Woodward, and most of 
them are vastly inferior. It is gratifying to state, however, that at the present 
time many original papers are partly or wholly illustrated by photo-micrographs, 
and no country has produced works with photo-micrographic illustrations superior 
to those in "Wilson's Atlas of Fertilization and Karyokinesis" and "Starr's Atlas 
of Nerve Cells," issued by the Columbia University Press. — 

In passing the writer would like to pay a tribute to Mr. W. H. Walmsley who 
has labored in advancing photo-micrography for the last twenty years. His con- 
venient apparatus and abundant experience have been placed freely at the com- 
mand of every interested worker, ;and many a beginner has been helped over 
difficulties by him. His last contribution in "International Clinics," vol. i. ser. 
11, 12, is encouraging in the highest degree both for its matter and for the 
illustrations. 




Fig. 1 S3. Zeiss' Vertical Photo-micro- 
graphic Camera. A. Set screw holding the 
rod (S ) in any desired position. P, O. Set 
screws by which the bellows are held in place. 
B. Stand with tripod base in which the sup- 
porting rod (S) is held. This rod is now 
graduated in centimeters and is a ready 
means of determining the length of the cam- 
era. M. Mirror of the microscope . L. The 
sleeve serving to make a light-tight connection 
betzveen the camera and microscope . O. The 
lower end of the camera. R. The upper end 
of the camera where the focusing screen and 
plate holder are situated. {From Zeiss' Photo- 
micrographic Catalog). 



As the difficulties of photo-micrography are so much greater than of ordinary 
photograph}^, the advice is almost universal that no one should try to learn photo- 
graphy and'photo-micrography at the same time, but that one should learn the 



CH. VIZI] PHOTO-MICROGRAPHY 223 

processes of photography by making portraits, landscapes, copying drawings, etc., 
and then when the principles are learned one can take up the more difficult subject 
of photo-micrography with some hope of success. 

The advice of Sternberg is so pertinent and judicious that it is reproduced : 
"Those who have had no experience in making photo-micrographs are apt to ex- 
pect too much and to underestimate the technical difficulties. Objects which 
under the microscope give a beautiful picture, which we desire to reproduce by 
photography may be entirely unsuited for the purpose. In photographing with 
high powers it is necessary that the objects to be photographed be in a single plane 
and not crowded together and overlying each other. For this reason photograph- 
ing bacteria in sections presents special difficulties and satisfactory results can only 
be obtained when the sections are extremely thin and the bacteria well stained. 
Even with the best preparations of this kind much care must be taken in selecting 
a field for photography. It must be remembered that the expert microscopist, in 
examining a section with high powers, has his finger on the fine adjustment screw 
and focuses up and down to bring different planes into view. He is in the habit of 
fixing his attention on the part of the field which is in focus and discarding the 
rest. But in a photograph the part of the field not in focus appears in a promi- 
nent way which mars the beauty of the picture." 

APPARATUS FOR PHOTO-MICROGRAPHY 

\ 362. Camera. — For the best results with the least expenditure of time one of 
the cameras especially designed for photo-micrography is desirable but is not by 
any means indispensable fordoing good work. An ordinary photographic camera, 
especiallj T the kind known as a copying camera, will enable one to get good results, 
but the trouble is increased, and the difficulties are so great at best, that one would 
do well to avoid as many as possible and have as good an outfit as can be afforded 
(Figs. 1S4, 192). 

The first thing to do is to test the camera for the coincidence of the plane occu- 
pied by the sensitive plate and the ground glass or focusing screen. Cameras even 
from the best makers are not always correctly adjusted. By using a straight edge 
of some kind, one can measure the distance from the inside or ground side of the 
focusing screen to the surface of the frame. This should be done all around to see 
if the focusing screen is equally distant at all points from the surface of the 
frame. If it is not it should be made so. When the focusing screen has been ex- 
amined, an old plate, but one that is perfectly flat, should be put into the plate 
holder and the slide pulled out and the distance from the surface of the plate 
holder determined exactly as for the focusing screen. If the distance is not the 
same the position of the focusing screen must be changed to correspond with that 
of the glass in the plate holder, for unless the sensitive surface occupies exactly 
the position of the focusing screen the picture will not be sharp, no matter how 
accurately one may focus. Indeed, so necessary is the coincidence of the plane of 
the focusing screen and sensitive surface that some photo-micrographers put the 
focusing screen in the plate holder, focus the image and then put the sensitive 
plate in the holder and make the exposure (Cox). This would be possible with 
the older forms of plate holders, but not with the double plate holders mostly used 
at the present day. 



224 PHOTO-MICROGRAPHY [CH. VIII 

\ 362a. Size of Camera. — The majority of photo-micrographs do not exceed 8 
centimeters in diameter and are made on plates 8 x 1 1 , 10 x 13 or 13 x iS centimeters 
(3#x4?4f in., 4x5 in., or 5x7 in.). Most of the vertical cameras are for plates not 
exceeding 10 x 13 centimeters (4x5 in. ) but Zeiss' new form will take plates 21 x 21 
centimeters (S^xS 1 ^ in.). 

\ 363. Work Room. — It is almost self-evident that the camera must be in 
some place free from vibration. Frequently a basement room where the camera 
table may rest directly on the cement floor or on a pier is an excellent situation. 
Such a place is almost necessary for the best work with high powers. For those 
living in cities, a time must also be chosen when there are no heavy vehicles 
moving in the streets. For less difficult work an ordinary room in a quiet part of 
the house or laboratory building will suffice. 

\ 364. Arrangement and Position of the Camera and the Microscope. — For 
much of photo-micrography a vertical camera and microscope are to be preferred 
(Fig. 1S4). Excellent arrangements were perfected long ago, especially by the 
French. (See Moitessier. ) 

Vertical photo-micrographic cameras are now commonly made, and by some 
firms only vertical cameras are produced. They are exceedingly convenient, and 
do not require so great a disarrangement of the microscope to make the picture as 
do the horizontal ones. Van Heurck advises their use, then whenever a structure 
is shown with especial excellence it is photographed immediately. The variation 
in size of the picture is obtained by the objective and the projection ocular rather 
than by length of bellows (see below Fig. 184). It must not be forgotten, how- 
ever, that penetration varies inversely as the sqtiare of the power, and only in- 
versely as the numerical aperture (# 34), consequently there is a real advantage in 
using a low power of great aperture and a long bellows rather than an objective of 
higher power with a short bellows. A horizontal camera is more convenient for use 
with the electric light also (Fig. 192). 

For convenience and rapidity of work a microscope with mechanical stage is 
very desirable. It is also an advantage to have a tube of large diameter so that 
the field will not be too greatly restricted (Fig. 189). In some microscopes the 
tube is removable almost to the nose-piece to avoid interfering with the size of the 
image. The substage condenser should be movable on a rack and pinion. The 
microscope should have a flexible pillar for work in a horizontal position. While it 
is desirable in all cases to have the best and most convenient apparatus that is 
made, it is not by any means necessary for the production of excellent work. A 
simple stand with flexible pillar and good fine adjustment will answer. 

\ 365. Objectives and Oculars for Photo-Micrography. — The belief is almost 
universal that the apochromatic objectives are most satisfactory for photography. 
They are employed for this purpose with a special projection ocular. Two very 
low powers are used without any ocular (Fig. 180). Some of the best work 
that has ever been done, however, was done with achromatic objectives (work of 
Woodward and others). One need not desist from undertaking photo-micrography 
if he has good achromatic objectives. From a somewhat extended series of ex- 
periments with the objectives of many makers the good modern achromatic ob- 
jectives were found to give excellent results when used without an ocular. Most 
of them also gave good results with projection oculars, although it must be said 
that the best results were obtained with the apochromatic objectives and projec- 




Fig. 184. — Vertical photo- 
micrographic camera,screen and 
small table. The table is about 
45 centimeters high and in the 
legs are large screw eyes for 
leveling screws. The operator 
can stand on the floor and per- 
form all the necessary opera- 
tions, and in adjusting the mi- 
croscope can sit on a low stool. 

The screen is of zinc and 
has two heavy lead feet to hold 
it steady. Near the lower left 
hand corner of the screen is an 
aperture for the light to shine 
through upon the mirror. This 
opening is closed by a black slide 
which is just balanced so that it 
stays in any position. In mak- 
ing the exposure it is raised 
sufficiently to admit the light to 
the mirror, but the stage is left 
in shadow. This screen shades 
the microscope and the face of 
the operator. ( Trans. Amer* 
Micr. Soc. 1 go 1. ) 



226 



PHO TO-MICROGRAPHY 



[CH. VIII 



tion oculars. It does not seem to require so much skill to get good results with 
the apochromatics as with the achromatic objectives. The majority of photo- 
micrographers do not use the Huygenian oculars in photography, although excel- 
lent results have been obtained with them. An amplifier is sometimes used in 
place of an ocular. Considerable experience is necessary in getting the proper 
mutual position of objective and amplifier. The introduction of oculars 
especially designed for projection, has led to the discarding of ordinary oculars 
and of amplifiers. However the projection oculars of Zeiss restrict the field very 
greatly, hence the necessity of using the objective alone for large specimens.* 

Fig. 185. Projection Oculars with section 
removed to show the construction. Below are 
shown the tipper end with graduated circle to 
indicate the amount of rotation found necessary 
to focus the diaphragm on the screen. No. .?, 
No. 4.. The numbers indicate the amount the ■ 
ocular magnifies the image formed by the ob- 
jective as with the compensation oculars. {Zeiss' 
Catalog. ) 

\ 366. Difference of Visual and Actinic 
Foci. — Formerly there was much difficulty ex- 
perienced in photo-micrographing on account 
of the difference in actinic and visual foci. 
Modern objectives are less faulty in this respect 
and the apochromatics are practically free 
from it. Since the introduction of orthochromatic or isochromatic plates and, in 
many cases the use of colored screens, but little trouble has arisen from differences 
in the foci. This is especially true when mono-chromatic light and even when 
petroleum light is used. In case the two foci are so unlike in an objective, it 
would be better to discard it for photography altogether, for the estimation of the 
proper position of the sensitive plate after focusing is only guess work and the 
result is mere chance. If sharp pictures cannot be obtained -with an objective 
when petroleum light and orthochromatic plates are used the fault may not rest 
with the objective but with the plate holder and focusing screen. They should be 
very carefully tested to see if there is coincidence in position of the focusing 
screen and the sensitive film as described in \ 362. 

\ 367. Apparatus for Lighting. — For low power work (35 mm. and longer 
focus) and for large objects, some form of bull's eye condenser is desirable 
although fairly good work may be done with diffused light or lamp-light reflected 
by a mirror. If a bull's eye is used it should be as nearly achromatic as possible. 
The engraving glass shown in Fig. 188 answers well for large objects. For smaller 




*A comparative study both with projection oculars, and without an ocular 
was made with the achromatic objective 25 mm. (1 inch), 18 mm. (finch), 5 
mm. (A to \ inch) and 2 mm. (jV inch) homogeneous immersion of the Bausch & 
Lomb Optical Co. ; Gundlach Optical Co. ; Leitz ; Reichert ; Winkel, Zeiss and the 
Spencer Lens Co. Good results were obtained with all of these objectives both 
with and without projection oculars. 



CH. VIII} 



PHO TO-MICROGRAPHY 



227 



objects a Steinheil lens combination gives a more brilliant light and one also more 
nearly achromatic. For high power work all are agreed that nothing will take the 
place of an achromatic condenser. This may be simply an achromatic condenser 
but preferably it should be an apochromatic condenser. Whatever the form of the 
condenser it should possess diaphragms so that the aperture of the condenser may 
be varied depending upon the aperture of the objective. For a long time objec- 
tives have been used as achromatic condensers, and they are very satisfactory, 
although less convenient than a special condenser whose aperture is great enough 
for the highest powers and capable of being reduced by means of diaphragms to the 
capacitv of the lower objectives. It should also be capable of accurate centering 
(§81). 




Fig. 186. Arrangement for Artificial Illumination. 

1. Lamp with metal chimney, easily made by rolling up some ferrotype plate 
and making a slit-like opening in one side. This opening should be covered by an 
oblong cover-glass. A glass slide, being of considerable thickness, breaks too easily. 
The lamp should have a wick about 40 mm. wide, so that the thickness of the flame , 
if taken edgewise, will give an intense light. A wide flame also enables one to get 
a larger image of the flame, and thus to illuminate a larger object than as though a 
small flame was used. 

2. Bull's-eye condenser on a separate stand. The engraving glass shown in 
Fig. 188, or the tripod magnifier {Fig. 172) answers fairly . The Steinheil lenses 
are still better. 

3. Screen showing image of the flame inverted. 

The lamp and bulls-eye stand are on blocks with screw-eyes as leveling screws. 

I 368. Objects Suitable for Photo-micrographs. — While almost any large ob- 
ject may be photographed well with the ordinary camera and photographic objec- 
tive, only a small part of the objects mounted for microscopic study can be photo- 
micrographed satisfactorily. Many objects that give beautiful images when look- 
ing into the microscope and constantly focusing with the fine adjustment, appear 
almost without detail on the screen of the photo-micrographic camera and in the 
photo-micrograph. 



228 



PHO TO-MICROGRAPHY 



[CH. VUI 




Fig. 187. Adjustable lens holder. This lens holder will take magnifiers of 
various sizes, and from its adjustable mechanism is very convenient for dissecting \ 
or for holding a Steinheil and other lenses for illumination ( The Bausch & Lomb 
Opt. Co.). 




Fig. 188. Engraving glass to 
serve as a condenser and for a dis- 
secting lens. {Bausch & Lomb Opt. 
Co.) 



If one examines a series of photo-micrographs the chances are that the greater 
number will be of diatoms, plant sections or preparations of insects. That is, they 
are of objects having sharp details and definite outlines, so that contrast and defi- 
niteness may be readily obtained. Stained microbes also furnish favorable objects 
when mounted as cover-glass preparations. 






CH. VIII} PHOTO-MICROGRAPHY 229 

Preparations in animal histology must approximate as nearly as possible to the 
conditions more easily obtained with vegetable preparations. That is, they must 
be made so thin and be so prepared that the cell outlines will have something of 
the definiteness of vegetable tissue. It is. useless to expect to get a clear photo- 
graph of a section in which the details are seen with difficulty when studying it 
under the microscope in the ordinary way. 

Many sections which are unsatisfactory as wholes, may nevertheless have parts 
in which the structural details show with satisfactory clearness. In such a case the 
part of the section showing details satisfactorily should be surrounded by a delicate 
ring by means of a marker (see Figs. 61-66). If one's preparations have been 
carefully studied and the special points in them thus indicated, they will be found 
far more valuable both for ordinary demonstration and for photography. The 
amount of time saved by marking one's specimens can hardly be overestimated. 
The most satisfactory material for making the rings is shellac colored with lamp- 
black. 

Ten years ago many histologic preparations could not be satisfactorily photo- 
graphed. But now with improved section cutters, better staining and mounting 
methods, and with the color screens (§ 356) and isochromatic plates (§355) almost 
any preparation which shows the elements clearly when looking into the micro- 
scope can be satisfactoril} 7 photographed. Good photographs cannot, however, be 
obtained from poor preparations. 

I 369. Light. — The strongest available light is sunlight. That has the defect of 
not always being available, and of differing greatly in intensity from hour to hour, 
day to day and season to season. The sun does not shine in the evening when 
many workers find the only opportunity for work. Following the sunlight the 
electric light is the most intense of the available lights. Then come magnesium, 
acetylene, the lime light, the gas-glow or Wellsbach light, and lastly, petroleum 
light. The last is excellent for the majority of low and moderate power work. 
And even for 2 mm. homogeneous immersion objectives, the time of exposure is 
not excessive for many specimens (40 seconds to 3 minutes). This light is also 
cheapest and most available and has the advantage of being somewhat yellow, and 
therefore in many cases makes the use of a color screen unnecessary if one uses 
isochromatic plates. Acetylene light is excellent and may be used where the arc 
light is not available. 

A lamp with flat wick about 40 mm. ( \% in. ) wide has been found most gen- 
erally serviceable. For large objects and low powers the flame may be made 
large and the face turned toward the mirror. This will light a large field. For 
high powers the edge toward the mirror gives an intense light. The ordinary 
glass chimney answers well, especially where a metal screen is used as shown 
in Fig. 184. 

EXPERIMENTS IN PHOTO-MICROGRAPHY 

§ 370. The following experiments are introduced to show prac- 
tically just how one would proceed to make photo-micrographs with 
various powers, and be reasonably certain of fair success. If one con- 
sults prints or the published figures made directly from photo-micro- 
graphs it will be seen that, excepting the bacteria, the magnification 
ranges mostly between 10 and 150 diameters. 



230 PHOTO-MICROGRAPHY [CH VIII 

§371. Focusing Screen for Photo-Micrography. — Onecannot 
expect a picture sharper than the image seen on the focusing screen. 
Hence the greatest care must be taken in focusing. The general focus- 
ing may be done with the unaided eye on the ground glass, but for the 
final focusing a clear screen and a focusing glass must be used. (Figs. 
172, 173). See § 347. With the clear focusing screen one cannot at 
first see the image without using a focusing glass, but with a little ex- 
perience the aerial image may be seen as with the microscope (§ 54). 

§ 372. Photo-micrographs of 20 to 50 Diameters. — For pic- 
tures under 15 or 20 diameters it is better to use the camera for embryos 
with the objective in the end of the camera, and the special microscope 
stand for focusing (Fig. 175). 

For pictures at 25 to 50 diameters one may use the microscope with 
a low objective, 25 to 35 mm. equivalent focus, and no ocular (Fig. 
184). The object is placed on the stage of the microscope, and focused 
as in ordinary observation. If a vertical microscope is used the light 
from the petroleum lamp or other artificial light, is reflected upward by 
the mirror. It may take some time to get the whole field lighted 
evenly. Refer back to § 95 for directions. In some cases it may be 
advisable to discard the condenser and use the mirror only. For some 
purposes one will get a better light by placing the bull's eye or other 
condenser between the lamp and the mirror to make the rays parallel 
or even to make a sharp image of the lamp flame on the mirror. Re- 
member also that in many cases it is necessary to have a color screen 
between the source of light and the object (§ 356). 

For a horizontal camera it is frequently better to swing the mirror 
entirely out of the way and allow the light to enter the condenser 
directly or after traversing the bull's eye (Figs. 182, 186). If the ob- 
ject is small an achromatic combination like a Steinheil magnifier or an 
engraving glass is excellent (Fig. 188). When the light is satisfac- 
torj r as seen through an ordinary ocular, remove the ocular. 

(A) Photographing without an Ocular. — After the removal of the 
ocular put in the end of the tube a lining of black velvet to avoid re- 
flections. Connect the microscope with the camera, making a light- 
tight joint and focus the image on the focusing screen. One may make 
a light-tight connection by the use of black velveteen or more con- 
veniently by the Zeiss' double metal hood which slips over the end of 
the tube of the microscope, and into which fits a metal cylinder on the 
lower end of the camera (Figs. 184, 189, 183). In the last figure the 
connection has been made. 



CH. VI II-] 



PHO TO-MICROGRAPHY 



231 




Fig. 189. Zeiss' special photo-micrographic stand. It has a very large tube, 
a slow acting fine adjustment, mechanical stage and all appliances for the most sat- 
isfactory work. ( Cut loaned by Eimer and Amend). 

It will be necessary to focus down considerably to make the image 
clear. Lengthen or shorten the bellows to make the image of the de- 
sired size, then focus with the utmost care. In case the field is too 
much restricted on account of the tube of the microscope, remove the 
draw-tube. When all is in readiness it is well to wait for three to five 
minutes and then to see if the image is still sharply focused. If it has 
got out of focus simply by standing, a sharp picture could not be ob- 



232 PHOTO-MICROGRAPHY [CH. VIII 

Figs. 190-191. Fine tint, half-tone reproductions of photo-micrographs of sec- 
tions made by Mrs. Gage, to show the possibilities of photo-micrography with pho- 
tographic objectives and with low microscopic objectives without a projection ocular. 

1. Frontal section of the head of a large red Diemyctylus viridescens {red 
newt) at the level of the portae of the brain, magnified 10 diameters. Negative 
made with a Gundlach perigraphic objective of about go vim. equivalent focus. 

2. Frontal section of a larval Diemyctylus about 10 millimeters in length. 
Negative made with a Winkel objective of 22 millimeters equivalent focus ; no 
ocular. Magnified 50 diameters. {Mrs. Susanna Phelps Gage, the Wilder 
Quarter Century Book). 

tained. If it does not remain in focus, something is faulty. When 
the image remains sharp after focusing make the exposure. From 20 
to 60 seconds will usually be sufficient time with medium plates and 
the light as described. If a color screen is used it will require 40-300 
seconds, i. e., 2 to 5 times as long, for a proper exposure (§ 359). 

B. Photog7-aphing with a Projection Ocular. — If the object is small 
enough to be included in the field of a projection ocular (Fig. 185) use 
that for making the negative as follows : Swing the camera around so 
that it will leave the microscope free. Use an ordinary ocular, focus 
and light the object, then insert a projection ocular in place of the or- 
dinary one, and swing the camera back over the microscope. It is not 
necessary to use an ordinary ocular for the first focusing, but as its 
field is larger it is easier to find the part to be photographed. The 
first step is then to focus the diaphragm of the projection ocular 
sharply on the focusing screen. Bring the camera up close to the mi- 
croscope and then screw out the eye-lens of the ocular a short distance. 
Observe the circle of light on the focusing screen to see if its edges are 
perfectly sharp. If not, continue to screw out the eye lens until it is. 
If it cannot be made sharp hy screwing it out reverse the operation. 
Unless the edge of the light circle, i. e. , the diaphragm of the ocular, 
is sharp, the resulting picture will not be satisfactory. 

It should be stated that for the x 2 projection ocular the bellows 
of the camera must be extended about 30 or 40 centimeters or the 
diaphragm cannot be satisfactorily focused on the screen. The X 4 
projection ocular can be focused with the bellows much shorter. For 
either projection ocular the screen distance can be extended almost 
indefinitely. 

When the diaphragm is sharply focused on the screen, the micro- 
scope is focused as though no ocular were present, that is, first with the 
unaided eye then with the focusing glass. The exposure is also made 
in the same way, although one must have regard to the greater mag- 



CH. VIII} PHOTO-MICROGRAPHY 233 

nification produced by the projection ocular and increase the time ac- 
cordingly ; thus when the X4 ocular is used, the time should be at 
least doubled over that when no ocular is employed. The time will be 
still further increased if a color screen is used (§ 359). 

Zeiss recommends that when the bellows have sufficient length the 
lower projection oculars be used, but with a short bellows the higher 
ones. It is also sometimes desirable to limit the size of the field by 
putting a smaller diaphragm over the eye lens. This also aids in 
making the field uniformly sharp. 

§ 373. Determination of the Magnification of the Photo- 
Micrograph. — After a successful negative has been made, it is desirable 
and important to know the magnification. This is easily determined 
by removing the object and putting in its place a stage micrometer. 
If the distance between two or more of the lines of the image on 
the focusing screen is obtained with dividers and the distance meas- 
ured on one of the steel rules, the magnification is found by dividing 
the size of the image by the known size of the object (§ 154). If now 
the length of the bellows from the tube of the microscope is noted, say 
on a record table like that in section 360, one can get a close approxi- 
mation to the power at some other time by using the same optical com- 
bination and length of bellows. 

For obtaining the magnification at which negatives are made it is 
a great advantage to have one micrometer in half millimeters ruled 
with coarse lines for use with the lower powers, and one in o. 1 and 
0.01 millimeter ruled with fine lines for the higher powers. 

§ 374. Photo-Micrographs at a Magnification of 100 to 150 
Diameters. — For this, the simple arrangements given in the preceding 
section will answer, but the objectives must be of shorter focus, S to 3 
mm. It is better, however, to use an achromatic condenser instead of 
the engraving glass or the Steinheil lens. 

§ 375. Lighting for Photo-Micrography with Moderate and 
High Powers. — (100 to 2,500 diameters). No matter how good one's 
apparatus, successful photo-micrographs cannot be made unless the ob- 
ject to be photographed is properly illuminated. The beginner can do 
nothing better than to go over with the greatest care the directions for 
centering the condenser, for centering the source of illumination, and 
the discussion of the proper cone of light and lighting the whole field, 
as given on pp. 41-52. Then for each picture the photographer must 
take the necessary pains to light the object properly. An achromatic 
condenser is almost a necessity (§ 80). Whether a color-screen should 



234 PHOTO-MICROGRAPHY \_CH. VIII 

be used depends upon judgment and that can be attained only by ex- 
perience. In the beginning one may try without a screen, and with 
different screens and compare results. 

A plan used by many skilled workers is to light the object and the 
field around it well and then to place a metal diaphragm of the proper 
size in the camera very close to the plate holder. This will insure a 
clean, sharp margin to the picture. This metal diaphragm must be 
removed while focusing the diaphragm of the projection ocular, as the 
diaphragm opening is smaller than the image of the ocular dia- 
phragm. 

If the young photo-micrographer will be careful to select for his 
first trials, objects of which really good photo- micrographs have 
already been made, and then persists with each one until fairly good 
results are attained, his progress will be far more rapid than as if poor 
pictures of many different things were made. He should, of course, 
begin with low magnifications. 

§ 376. Adjusting the Objective for Cover-Glass. — After the 
object is properly lighted, the objective, if adjustable, must be cor- 
rected for the thickness of cover. If one knows the exact thickness 
of the cover and the objective is marked for different thicknesses, it is 
easy to get the adjustment approximately correct mechanically, then 
the final corrections depend on the skill and judgment of the worker. 
It is to be noted too that if the objective is to be used without a projec- 
tion ocular the tube-length is practically extended to the focusing screen 
and as the effect of lengthening the tube is the same as thickening the 
cover- glass, the adjusting collar must be turned to a higher number 
than the actual thickness of the cover calls for (see § 103). 

§ 377. Photographing Without an Ocular. — Proceed exactly 
as described for the lower power, but if the objective is adjustable make 
the proper adjustment for the increased tube-length (§ 103). 

§ 378. Photographing with a Projection Ocular. — Proceed as 
described in § 372 B, only in this case the objective is not to be adjusted 
for the extra length of bellows. If it is corrected for the ordinary 
ocular, the projection ocular then projects this correct image upon the 
focusing screen. 

§ 379. Photo-Micrographs at a Magnification of 500 to 2000 
Diameters. — For this the homogeneous immersion objective is em- 
ployed, and as it requires a long bellows to get the higher magnifica- 
tion with the objective alone, it is best to use the projection oculars. 



CH. VIII} PHOTO-MICROGRAPHY 235 

For this work the directions given in § 372 B must be followed with 
great exactness. The edge of the petroleum lamp flame is sufficient to 
fill the field in most cases. With many objects the time required with 
good lamp light is not excessive ; viz., 40 seconds to 3 minutes. The 
reason of this is that while the illumination diminishes directly as the 
square of the magnification, it increases with the increase in numerical 
aperture, so that the illuminating power of the homogeneous immersion 
is great in spite of the great magnification (§ 34). 

For work with high powers a stronger light than the petroleum 
lamp is employed by those doing considerable photo-micrography. 
Good work may be done, however, with the petroleum lamp. 

It may be well to recall the statement made in the beginning, that 
the specimen to be photographed must be of especial excellence for all 
powers. No one will doubt the truth of the statement who undertakes 
to make photo-micrographs at a magnification of 500 to 2000 diameters. 

If one has a complete outfit with electric arc light (Fig. 192) the 
time required for photographing objects is much reduced, i. e. ranging 
from 1 to 20 seconds even with a color screen. As the light is so in- 
tense with the arc light it is necessary to soften it greatly for focusing. 
Several thicknesses of ground glass placed between the lamp and the 
microscope will answer. These are removed before taking the nega- 
tive. It is well also to have a water bath on the optical bench to ab- 
sorb the heat rays. This should be in position constantly (see Ch. IX). 

§380. Use of Oculars in Photo-Micrography. — There is much 
diversity of opinion whether or not the ordinary oculars used for ob- 
servation should be used in photographing. Excellent results have 
been obtained with them and also without them. 

For great magnification Zeiss recommends the use of the compen- 
sation oculars with the apochromatics. 

The Zeiss projection oculars may be used with achromatic objectives 
of large aperture as well as with the apochromatics. 

PHOTOGRAPHING OPAQUE OBJECTS AND METALLIC SURFACES 
WITH A MICROSCOPE 

All of the objects cansidered in the first part of this chapter are opaque and 
some of them were to be photographed somewhat larger than natural size. To 
meet the needs of modern work, especially with metals and alloys one must be 
able to examine and photograph prepared surfaces at magnifications ranging from 
five or ten to five hundred or more diameters. 

\ 381. Microscope for Opaque Objects. — If one does not need to magnify 
more than about 100 diameters, any good microscope will answer. For the higher 



236 



PHO TO -MICROGRA PHY 



\CH. VIII 




CH. VI II] 



PHO TO-MICROGRA PH Y 



237 



Fig. 192. Buxton's Photo-Micrographic outfit for use with the arc tight. 
{Jour. Ap. Microscopy, 1901, p. 1367). {Cut loaned by the Bausch & Lomb. Opt. 
Co.) As will be seen from the figure this apparatus is for work in the horizontal 
position. The optical bench cotitaining the microscope, water bath, color screen and 
the electric light, swings sidewise sufficiently for the operator to arrange the speci- 
men exactly as desired. It then swings back into position and is joined to the 
camera. This is in two sections for either a short or long bellows. This seems to 
be the most convenient of all the expensive outfits for photo-micrography . 

powers it is far more convenient to employ a special microscope for metallography 
(micro-metalloscope.) (German, Metallmikroskop ; French, Microscope pour 
l'etude des surfaces metalliques et des objets opaque). (Fig. 193.' 




Fig. 193. Special microscope of the Boston Testing Laboratories for the study 
and photography of metals and alloys {\ 381). {Cut loaned by the Boston Testing 
Laboratories. ) 



238 PHOTO-MICROGRAPHY [CH VIII 

Such a microscope has the following general characters : The stage is mova- 
ble up and down with rack and pinion, it is rotary and more or less mechanical by 
means of centering screws. With some at least the stage may be removed 
entirely. No substage condenser is present, and a mirror is only present for occa- 
sional transparent objects. A revolving nose-piece is not so good as the objective 
changers. 

\ 382. Illumination of Opaque Objects. — (A) for 25 to 100 diameters. The 
directions of Mr. Walmsley are excellent (Trans. Amer. Micr. Soc, 1S9S, p. 191). 
"Altogether the best light for the purpose is diffused daylight. Proper lighting is 
more easily obtained with a vertical camera. An even illumination avoiding deep 
shadows is preferable in most cases and is more easily attained with the object in 
a horizontal position. For many objects it is better not to use a bull's eye or any 
form of condenser but for others the condenser may be needed, but when the con- 
denser is used one must avoid too much glare. The now little used parabolic re- 
flector and L,ieberkiihn serve well in many cases, but he adds "the majority yield 
better results under the most simple forms of illumination," z. e. , with the dif- 
fused light from the window. This has been the experience of the writer also. 

In case diffused daylight is employed the camera should be near a good sized 
window, and the object should be somewhat below the window ledge so that the 
illumination is partly from above and from the side. (This is easily attained with 
the small table and vertical camera shown in Figs. 175, 184). The vertical illum- 
inator is advantageous for these powers also. See (B. ). 

(B) For 100 to 500 diameters, — For the magnifications above 50 it is desirable 
and for those above 100 it is necessary to use some form of "vertical illuminator," 
that is some arrangement by which the light is reflected down through the objec- 
tive upon the object, the objective acting as a condenser, and from the object 
back through the objective and ocular to the eye of the observer. This is accom- 
plished in two ways : 

( 1 ) By means of a small speculum-metal mirror in the tube of the micro- 
scope. This is set at an angle of 45 degrees and the light thrown into the tube 
upon it is reflected straight down through the objective upon the object. The 
speculum metal being opaque cuts out a part of the light. Instead of a metal 
mirror a circular disc of glass is now more frequently used. This allows the 
major part of the light reflected from the object, up through the objective to 
reach the eye. 

(2) By means of a small glass 45 degree prism inserted into the side of the 
objective or of a special adapter. The light is from the side of the microscope, and 
is reflected by the prism straight down through the objective upon the object as 
before.* 



*The idea of the vertical illuminator apparently originated with Hamilton L. 
Smith. He used the metal- reflector. Beck substituted a cover-glass and Powell 
and Zealand a disc of worked glass; i. e. glass that had been carefully polished 
and leveled on the two sides. Carpenter-Dallinger, pp. 336-338. 

The use of the prism with the objective is due to Tolles (see Jour. Roy. Micr. 
Soc, vol. iii, 1880, pp. 526, 574). 

In Zeiss' catalog the prism form is figured. In the catalog of Nachet both the 
glass disc and the prism forms are figured. 



CH. VHP] PHOTO-MICROGRAPHY 239 

For both these devices uncovered objects are most successful or if the object 
is covered it must be in optical contact with the cover-glass. Naturally good re- 
flecting surfaces like the rulings on polished metal bars give most satisfactory 
images, hence this method of illumination is especially adapted to micro-metal- 
lography. Indeed, without some such adequate method of illumination the study 
of metals and alloys with high powers would be impossible. So successful is it 
that oil immersion objectives may be used. (Carpenter-Dallinger, pp. 335-338). 

§ 383. Light for the Vertical Illuminator. — For moderate 
powers one may place the microscope in front of a window, or one may- 
use a petroleum or gas lamp. For the higher powers acetylene or 
preferably the electric arc light is used. In either case it may be neces- 
sary to soften the light somewhat either by a color screen or by some 
ground glass. The light should be concentrated upon the exposed end 
of the prism or into the hole leading to the glass disc. Both the prism 
and the disc should be adjustable for different objectives and different 
specimens. The cone of light, especially with the electric arc light, 
should be enclosed in a hollow metal or asbestos cone to avoid the glare 
in the eyes of the operator, and it may be necessary to soften the light 
with ground glass before attempting to focus and arrange the speci- 
men. This ground glass would in most cases be removed before mak- 
ing the exposure (§ 379). 

With the electric light and for long exposure or observation a 
water bath to absorb the heat rays will be necessary to avoid injuring 
the lenses. (See also under projection in the next chapter). 

As it is somewhat difficult to adjust the light in a way to give the 
best effect, one can see the advantage of the adjustment for raising and 
lowering the stage. This will serve for all but the finest focusing, and 
thus avoid moving the tube for focusing enough to throw the lighting 
out of adjustment. It might be advantageous to have a fine adjust- 
ment on the stage also. 

? 3S4. Mounting of Objects. — For observation only and with low powers, 
the objects may be mounted either in a liquid or dry as seems best. There should 
be a black background for most objects, then light will reach the eye only from 
the object. A light background is sometimes desirable, especially where one cares 
only for outlines. 

1 385. Preparation of Metallic Surfaces. — In the first place a flat face is 
obtained by grinding or filing, and then this is polished. For polishing, finer 
and finer emery or other polishing powders are used, (rouge or diamantine, 
or specially prepared alumnina, etc). The aim is to get rid of the scratches so 
that the surface will be smooth and free from lines. 

\ 386. Etching. — After the surface is polished it should be etched with some 
substance. This etching material will corrode the less resistant material, the 
edges of crystals, etc., so that the structure will appear clearly. For etching, 



240 PHOTO-MICROGRAPHY [CH. VI/I 

tincture of iodine, nitric acid in various degrees of strength, hydrochloric acid, 
etc., are used or one may use electricity, the metal being immersed in an indiffer- 
ent liquid. See numerous articles in the Metallographist for methods and micro- 
graphs. 

After the etching, the surface should be washed well with water to remove the 
etcher. L,e Chatelier recommends that the etched surface when dry be coated 
with a very thin coating of collodion to avoid tarnishing. The preparation will 
then last for several months untarnished. 

\ 387. Mounting the Preparations of Metal. — In order to get a satisfactory 
image the flat, polished and etched face should be at right angles to the optic axis. 
For preliminary observation one can approximate this by mounting the specimen 
on a piece of beeswax. (Behrens). Very elaborate arrangements of the stage 
have also been devised (Reichert). A simple and effective device is shown in Fig. 
193 in which the specimen is held against the under side of the plane face of the 
stage attachment. Rubber bands answer well to support the metal, and only one 
side need be flat. 

\ 388. Photographing Opaque Objects. — The general directions given in \ 347 
should be followed with the necessary modifications. The time of exposure is 
usually considerably greater with opaque objects than with transparent ones. 
Very few such objects can be photgraphed in less than 30 seconds, even with day- 
light For metallic surfaces and magnifications of 100, 150, 250 to 500, with the 
electric arc light as illuminant the time required for favorable objects is 1, 2, 
4 and 7 seconds ; with the Wellsbach lamp the time is 5, 10, 30 and 60 minutes 
(Sauveur). 




Fig. 194. Rack for drying negatives 
{Rochester Opt. Co). 



Fig. 194. 

References to Ch. VIII. 

See the works and journals dealing with photography. 

For Photo-Micrography see Pringle, Bousfield, Neuhauss, Sternberg, Francotte 
and the special catalogs on photo-micrography and projection issued by the great 
opticians. The Journal of the Royal Microscopical Society and of the Quekett 
Micr. Club ; Zeit. wiss. Mikroskopie ; the Trans Amer. Micr. Soc. ; the Amer. 
Monthly Micr. Journal ; the Journal of Applied Microscopy. 

For the photography of metallic surfaces, see the various journals of engineer- 
ing and metallurgy, but especially Sauveur's journal, the Metallographist, begun 
in 1898. 



CH. Vf/I] PHOTO-MICROGRAPHY 241 

ENLARGEMENTS \ LANTERN SLIDES ; PHOTOGRAPHING 
BACTERIAL CULTURES 

\ 389. Enlargements. — As a low power objective has greater depth of focus 
or penetration than a higher power ($ 34), it is desirable in many cases to make a 
negative of an object with considerable depth at a low magnification, and then 
to enlarge this picture to the desired size. As a rule negatives will not bear an 
enlargement of more than five diameters. 

For this work the camera shown in Fig. 18 r is excellent, and the special mi- 
croscope stand shown in this figure and in Fig. 175 serves to enable one to get a 
very exact focus. 

One must select an objective for the enlargement with a field of sufficient size 
to cover the part of the negative to be enlarged. An objective of 60 to 100 mm. 
focus will answer in most cases. 

For the illumination the camera can be elevated against the sky, or artificial 
light may be used. It is not easy to light so large a surface evenly by artificial 
light. 

(A) Enlargeme7it 011 Bromide Paper. — For this the negative is put in place 
and by pulling out the bellows the proper amount, one gets the right magnifica- 
tion. Focus now as for any other object, using the fine adjustment and focusing 
glass. 

For great exactness one must put a clear glass in the plate holder and focus 
on the surface away from the objective. Then place the bromide paper on this 
clear glass and put another over it to hold it flat against the first plate of glass. 
The sensitive surface will then be in the exact plane of the focus and the picture 
will be sharp. 

For the development and subsequent treatment of the paper, follow the 
directions of the makers. 

(B) Enlargement on a Glass Plate.— One may proceed in enlarging as for 
making lantern slides and make a positive on a glass plate. If it is then desired 
to get a negative for printing, place this positive on the microscope stand and 
make a negative from it as if it were an object. Or one may make a contact im- 
pression as is frequently done in lantern slide making. By this method one must 
make three separate pictures, (1) the original photo-micrographic negative ; (2) 
the enlarged positive from this ; (3) a negative from the enlarged positive. With 
this negative one may print as from the original negative. 

I 390. Lantern Slides from Negatives. — In preparing lantern slides from 
photo-micrographic or ordinary negatives one may use the contact method, or the 
camera. With the camera one can enlarge or reduce to suit the particular case. 
The camera and special microscope stand shown in Fig. 181 are admirable for the 
purpose. For lantern slide work a photographic objective is used and the cone 
for enlargement removed. One may put the objective in the front of the camera 
or in the middle segment, making use of the little side door. 

\ 391. Photographing Bacterial Cultures in Petri Dishes. — For the successful 
photographing of these cultures dark ground illumination is employed on the 
principal stated in § 92. That is the preparation is illuminated with rays so 
oblique that none can enter the objective. These striking the culture are reflected 



242 PHOTO-MICROGRAPHY [ CH. VIII 

into the objective. The clear gelatin around the growth or colonies does not 
reflect the light and therefore the space between the colonies is dark. 

For supporting the Petri dishes a hole is made in a front board for the camera. 
This hole is slightly larger than the dish. Over it is then screwed or nailed a 
rubber ring slightly smaller than the Petri dish. This will stretch and receive the 
dish, and grasp it firmly so that it is in no danger of falling out when put in a verti- 
cal position. If the camera has two divisions like the one shown the board with 
the Petri dish is put in the front of the camera, and the objective in the middle 
division through the side door. Otherwise the board holding the Petri dish must 
be on a separate support. 

The illumination is accomplished by the use of two electric lamps with conical 
shades. (The cheap tin shades with white enamel paint on the inside are good). 
The lamps are placed at the sides so that a bright light is thrown on the culture, 
but at such an angle that none of it enters the objective directly. 

A piece of black velveteen is placed 10 to 20 cm. beyond the culture. This 
prevents any light from being reflected through the clear gelatin to the objective. 
Unless some such precaution were taken the background would be gray instead of 
black. 

One may use daylight by putting the culture in a support just outside a win- 
dow, leaving the camera in the room. The rays from the sky are so oblique that 
they do not enter the objective. One must use a black non-reflecting background 
some distance beyond the dish as in using artificial light (Atkinson). 

\ 392. Photographing Bacterial Cultures in Test-Tubes. — Here the lighting 
is as in the preceding section, but a great difficulty is found in getting good re- 
sults from the refraction and reflections of the curved surfaces. To overcome this 
one applies the principles discussed in $ 144, and the test-tubes are immersed in a 
bath of water or water and glycerin. The bath must have plane surfaces. Behind 
it is the black velvet screen, and the light is in front as for the Petri dishes. As 
suggested by Spitta it is well to employ a bath sufficiently thick in order that 
streak cultures may be arranged so that the sloping surface will all be in focus 
at once by inclining the test-tube. 



See the works on photo-micrography and photography for the details of lan- 
tern slide making. See for the Petri dishes and test-tubes, Atkinson, Botanical 
Gazette, xviii (1893), p. 333 ; Spitta, Photo-Micrography (1899), p. 26. 



CHAPTER IX 

CLASS DEMONSTRATIONS IN HISTOLOGY AND 
EMBRYOLOGY 



APPARATUS AND MATERIAL FOR THIS CHAPTER 

Demonstration microscopes, simple and compound (Figs. 195-196); Traveling 
microscope (Fig. 197-198); Indicator ocular (Fig. 199-201); Marker for putting 
rings around the parts of specimens to be demonstrated (Fig. 61); Projection mi- 
croscope (Fig. 207); Projection objectives (Fig. 211-212); Episcope (Fig. 214). 



DEMONSTRATION MICROSCOPES AND INDICATORS 

§ 393. Simple Microscope. — The simple microscope held in 
one hand and the specimen in the other, has alwa3^s been used for 
demonstration, but for class demonstration it is necessary to have mi- 
croscope and specimen together or the part to be observed by the class 
is frequently missed. Originally blocks of various kinds to hold both 
microscope and specimen were devised, but within the last few years 
excellent pieces of apparatus have been devised by several opticians for 
the purpose. The accompanying figure shows one of the best forms. 



Fig. 195. Simple Demonstra- 
tion Microscope of Leitz ( Wm. 
Krafft, N. Y. ) As shown in 
the figure this consists of a 
handle, a stage and a lens holder 
which slides up and down for 
focusing. F01 observation the 
student holds it up to the light. 




§ 394- 



Fig. 195. 

Compound Demonstration Microscope. — This was 
originally called a clinical or pocket microscope. It is thus described 
by Mayall in his Cantor Lectures on the history of the microscope : ' 'A 
small microscope was devised by Tolles for clinical purposes which 
seems to me so good in every way that I must ask special attention for 



244 



CLASS DEMONSTRA T10NS 



[ CH. IX 



it. The objective is screwed into a sliding tube, and for roughly focus- 
ing the sliding motion suffices ; for fine adjustment, the sheath is made 
to turn on a fine screw thread on a cylindrical tube, which serves also 
as a socket-carrier for the stage. The compound microscope is here 
reduced to the simplest form I have met with to be a really serviceable 
instrument for the purpose in view ; and the mechanism is of thor- 
oughly substantial character. I commend this model to the notice of 
our opticians." 




Fig. 196. Demonstration compound 
microscope of Leitz. Leitz now furnishes 
a fine adjustment in the form of an inter- 
mediate piece between the objective and 
the tube. This has in it a screw which is 
turned by a milled ring. For the object- 
ives employed it makes an efficient fine 
adjustment and renders it possible for each 
person to adjust the microscope slightly 
without endangering the loss of the field. 



Fig. 196. 

Since its introduction by Tolles many opticians have produced ex- 
cellent demonstration microscopes of this type, but most of them have 
not preserved a special mechanism for fine adjustment. With it one 
can demonstrate with an objective of 6 mm. satisfactorily. It has a lock 
so that once the specimen is in the right position and the instrument fo- 
cused it may be passed around the class. For observation it is only 
necessary for each student to point the microscope toward a window or 
a lamp. 



CH. IX} 



CLASS DEMONSTRA T10NS 



245 




Fig. 197. 

Fig. 197. Traveling microscope set tip for work (Leitz ; from Wm. 
Xrafft, N. Y). 

A modification of this clinical microscope was made by Zentmayer 
in which the microscope was mounted on a board and a lamp for illum- 
inating the object was placed at the right position. 



246 



CLASS DEMONSTRATIONS 



\_CH. IX 



§ 395. Traveling Microscope. — For many years the French 
opticians have produced most excellent traveling microscopes. The 
opticians of other countries have also brought out serviceable instru- 
ments. In the one here figured Mr. Leitz has combined in an admirable 
way a traveling microscope and a laboratory instrument. For the 
needs of the pathologist and sanitary inspector a microscope must pos- 
sess compactness and also the qualities which render it usable for nearly 
all the purposes required in a laboratory. This instrument is a type 
of such apparatus which has grown up with the needs of advancing 
knowledge. 




Fig. 198. 

Fig. 198. Tiaveling microscope folded up and in its case {Leitz ; from Wm. 
Kraffl, N. Y.). 



CH. IX] 



CLASS DEMONSTRATIONS 



247 



§ 396. Indicator or Pointer Ocular. — This is an ocular in 
which a delicate pointer of some kind is placed at the level where the 
real image of the microscope is produced. It is placed at the same 
level as the ocular micrometer, and the pointer like the micrometer is 
magnified with the real image and appears as a part of the projected 
image (§ 170). Bj t rotating the octdar or the pointer any part of the 
real image may be pointed out as one uses a pointer on a wall or black- 
board diagram. By means of the indicator eye-piece one can be cer- 
tain that the student sees the desired object, and is not confused by the 
multitude of other things present in the field. The method of its use 
is indicated in Fig. 201. This device has been invented manj^ times. 
It illustrates well the adage : "necessity is the mother of invention," 
for what teacher has not been in despair many times when trying to 
make a student see a definite object and neglect the numerous other 
objects in the field. So far as the writer has been able to learn, 
Quekett was the first to introduce an indicator ocular with a metal 
pointer which was adjustable and could be turned to any part of the 
field or wholly out of the field. See Fig. 199, § 126. 





Fig. 199. Fig. 200. Fig. 2or. 

Fig. 199. Indicator ocular with -metal pointer like the one devised by 
Quekett (Leitz ; catalog). 

Fig. 200. Indicator ocular with an eyelash {cilium) on the ocular diaphragm 
to serve as a pointer ( P) . This projects about half way across the diaphragm open- 
ing. On the opposite side are shown two rays from the microscope to indicate that 
the real image is formed at the level of the ocular diaphragm. 

Fig. 20 1 . Field of the microscope with a mammalian blood preparation to show 
the use of the indicator (P) for pointing out a white blood corpuscle. 



CLASS DEMONSTATIONS 



[CH. IX 



It is not known who adopted the simple device of putting the tip 
of a cat's whisker or an eye-lash on the diaphragm of the ocular as 
shown in Fig. 200. This ma} 7 be done with any ocular, positive or 
negative. One may use a little mucilage, Canada balsam or any 
other cement, and stick the eyelash on the upper face of the diaphragm 
so that it projects about half way across the opening. When the eye- 
lens is screwed back in place the hair should be in focus. If it is not 
screw the eye-lens out a little and look again. If it is not now sharp, the 
hair is a little too high and should be depressed a little. If it is less 
distinct on screwing out the ocular it is too low and should be elevated. 
One can soon get it in exact focus. Of course it may be removed at 
any time. 

§ 397- — Marking the Position of Objects. — In order that one 
may prepare a demonstration easily and certainly in a short time the 
specimens to be shown must be marked in someway. A very efficient 
and simple method is to put rings of black or colored shellac around 
the part to be demonstrated. For this the Marker, Fig. 61-62, is 
employed as described on p. 66. 





©©#©#© 
© ©(g)® © ® 
$ ® m ® ® • 





Fig. 202. 

Fig. 202. Ring around one of the sections of a series for demonstrating some 
organ especially well. 




Fig. 203. Figure of a microscopical 
preparation with a ring around a small 
part to show the position of some structural 
feature. 



CH. IX] PROJECTION MICROSCOPE 249 

PROJECTION MICROSCOPE 



One of the most useful and satisfactory means at the disposal of 
the teacher of Microscopic Anatomy and Embryology for class demon- 
strations is the Projection Microscope. With it he can show two 
hundred as well as one person the objects which come within the range 
of the instrument. 

It is far more satisfactory than microscopic demonstrations, for 
w r ith the projection microscope the teacher can point out on the screen 
exactly the structural features and organs which he wishes to demon- 
strate, and he can thus be certain that the students know exactly what 
is to be studied. Unless one employs a pointer ocular (Fig. 201), 
there is no certainty that the student selects from the multitude of things 
in the microscopic field the one which is meant by the teacher. Like 
all other means, however, the projection microscope is limited. With 
it one can show organs both adult and embryonic, and the gen- 
eral morphology. For the accurate demonstration of cells and cell 
structure the microscope itself must be used by each student personally. 

If no ocular is used a 3 mm. or }& inch objective is about as high a 
power as can beemplo3'ed in a room holding two hundred. Even with 
an oil immersion fair demonstrations can be made, but up to the pres- 
ent time really successful demonstrations are usually made with powers 
below 3 mm. rather than above. If projection oculars are used one 
can hardly go beyond an 8 mm. objective with real satisfaction. And 
finally it should be remembered that the Continuous Current Arc Lamp 
must be employed for satisfactory results. Alternating currents are 
wholly unsatisfactory for this purpose. 

§ 398. Projection Microscope. — This is an arrangement of the 
microscope so that an image of the object under the microscope is 
thrown upon a screen of some kind. The picture on the screen is 
looked at precisely as one looks at the pictures thrown on the screen 
by an ordinary magic lantern. Indeed the -projection microscope is a 
magic lantern with short focus objectives. One of the first uses of the 
microscope was to throw the images of various objects on a screen so 
that they could be seen by several persons at once, and the light used 
was sunlight. Hence those early projection microscopes were called 
solar or sun microscopes. If sunlight were available at all times and 
could be controlled, it would be universally employed ; but as it is not 



250 PROJECTION MICROSCOPE {CH. IX 

at all times available and whenever available a heliostat is needed to 
keep the light fixed in a given position, sunlight is practically dis- 
carded and the electric light is employed for illumination. 

§ 399. Parts of a Projection Microscope. — These are named 
in order, beginning with the electric lamp : See Figs. 192, 207-208. 

(1) An arc lamp with constant current and rheostat adjustable 
from about 8 to 20 amperes. 

(2) Lamp condenser. 

(3) Water bath for removing the heat rays. 

(4) Special achromatic condenser for high powers. 

(5) Large square stage with opening 6 centimeters in diameter. 

(6) Mechanical stage with wide range of movement. 

(7) A very wide tube for receiving the objectives and projection 
oculars. This tube to be connected with coarse and fine adjustment 
for focusing. 

All of these parts should be independent and adjustable so that 
any one of them can be adjusted or removed without interfering with 
the others. 

In accordance with the suggestion of Dr. Coplin all of the appa- 
ratus, including the mountings of the objectives should be dead black 
to avoid reflections. Reflections are so dazzling that the operator can- 
not properly focus the image on the screen. 

§ 400. The Arc Lamp for the Projection Microscope. — This 
should have the following characters : (a) The carbons should be 
tilted so that the crater in the positive carbon is nearly vertical. It 
then sends the maximum number of rays to the lamp condenser. 
(J?) The lower carbon should be slightly in advance of the upper one 
as shown in Fig. 204 so that the crater in the positive carbon is on the 
front of that carbon (Figs. 204-205) that is it should be in position 
to throw the light toward the condenser. 

(V) The lamp must be adjustable vertically and horizontally, and 
the carbon holders must also be adjustable so that the carbons may be 
put in line from side to side and front to back. One cannot get satis- 
factory results unless all these adjustments are possible. 

Hand-feed and automatic lamps are both used for projection. The 
consensus of opinion among experts is that the hand-feed lamp is bet- 
ter for photography and for projection. That has been the experi- 
ence of the writer also. 

S 401. Starting the Lamp. — For starting the lamp with the 
hand-feed form it is necessary to bring the carbons in contact and then 



CH. IX] PROJECT/ON MICROSCOPE 251 

when the current is established to separate them slightly (1 to 2 mm.) 
in order to get a satisfactory light. The carbons may be brought in con- 
tact before turning on the current or afterwards. An automatic lamp 
will start as soon as the current is turned on, but here also the carbons 
must be slightly separated, or one must wait a short time for the car- 
bons to wear away before the best light is obtained. 

§ 402. Angle of the Carbons. — Originally arc lamps for use 
with the lantern had the carbons both vertical. The "projector" lamps 
used at sea, had, however, the carbons inclined at an angle of 30 or 40 
degrees from the vertical. Lewis Wright (Optical Projection, p. 163,) 
states that at his urgent request a projection arc lamp for micro-projec- 
tion was made with inclined carbons. Certain it is that all projection 
lamps have now the inclined carbons. The angle of inclination varies 
with different makers. The lamps furnished by Zeiss and Reichert 
with their apparatus has the carbons at 40 degrees from the vertical ; 
Behrens uses and recommends 45 degrees. Barnard and Carver (J. R. 
M. S., 1898, p. 170) found by a careful series of experiments that an 
angle of about 27 degrees gave the most satisfactory light. One firm 
( A.T. Thompson & Co. of Boston) arranges the carbons at right angles, 
the upper or positive carbon being horizontal. This gives good results. 
The purpose of the inclination is to turn the crater toward the con- 
denser (Fig. 205), for it is now appreciated that the arc proper gives 
very little light comparatively. One will appreciate this by studying 
the carbons projected on the screen as suggested in § 406. 

§ 403. Adjusting the Carbons. — In many arc lamps for projec- 
tion there is a metal shelf to show approximately where the two car- 
bons should meet. If one places the carbons in their holders so that 
the ends are at the level of this shelf they will be nearly in the correct 
position. 

The lower carbon should be slightly in advance of the upper one 
(Fig. 204). This will insure the formation of the crater on the side 
facing the condenser. 

§ 404. Length of the Arc. — It has been found by careful inves- 
tigation fCarhart, Ayrton) that the maximum brilliancy and efficiency 
of a continuous current arc lamp are obtained when the arc is about 1 
mm. long and the current is about at its maximum for the size of car- 
bon used. If the carbons are too far apart the light becomes purplish. 
If the current is too weak the lower carbon is blunt, while with a 
stronger current it becomes more pointed, and hides less light ; it also 
contributes a share of the illumination from its white tip (Fig. 205). 



252 



PROJECTION MICROSCOPE 



[CH. IX 



In order to see whether the carbons are in the most favorable posi- 
tion, Barnard and Carver (J. R. M. S.. 1898, p. 171) used a pin-hole 
camera at right angles to the carbons. This camera has a ground 
glass with cross lines to serve as guides in maintaining the proper 
position of the carbons. 




Fig. 204. 



Fig. 205. 



Figs. 204-205. Front and side views of the carbons of an arc light to give the 
best illumination. -\- and — indicate the positive and negative poles 

Fig. 204 is a side view showing the carbons in section at an angle of 30 degrees 
from the vertical and the negative ( — ) or lower carbon slightly in front of the pos- 
itive ( + ) or upper carbon. The carbons have soft cores. 

Fig. 205 is a front view of the carbons as seen projected on the screen with a 42 
mm. objective. It is a projection of the real image of the carbons formed by the 
special achromatic condenser next the object (§ 399, 4). This figure shows that the 
source of light is the crater in the positive ( -f ) or upper carbon ; it shows also that 
the lower carbon is slightly below the lower carbon as well as slightly in front. This 
avoids a shadow from the lower carbon. 

In the center of the crater is shown a slight shadow. This is due to the pit 
formed in the soft core of the carbon. 

% 405. Positive and Negative Carbon. — The mechanism of 
the lantern is arranged so that the upper carbon should be positive 
(+). In setting up the apparatus one may not be able to insert the 
wires correctly at first. All one has to do is to make the connections, 



CH. IX] PROJECTION MICROSCOPE 253 

turn on the current and observe which carbon is the more brilliant. As 
shown in figure 205 the brilliant carbon is at the positive pole. If now 
the upper carbon is brilliant the wires are properly connected ; but if 
the lower carbon is the brighter, the wires are inserted in the wrong 
binding posts and should be reversed. In observing the carbons when 
the current is on, one should use deeply colored glass to avoid injuring 
the eyes. Another excellent method is to turn the current off after a 
minute or two and look at the carbons directly. The one at the pos- 
itive pole will be red or white hot while the other will be black or very 
dull red. 

§406. Character of the Carbons and Steadiness of the Light. 
— One needs a steady light for photography and for projection. To hold 
the crater in one position and thus render the light steady, a softer 
core is placed in the carbons (Fig. 204). This serves as a guide, and 
the crater forms symmetrically around it when the carbons are in a 
proper position (Fig. 205). Every one who wishes to make a success, 
of micro-projection is urged to study the appearance of the carbons by 
using a low objective (35 to 65 mm.). The real image of the carbons 
formed by the achromatic condenser next the object (§ 399, 4) can be 
focused and thrown on the screen as if it were an object and one 
can study the crater. The image on the screen will be right side up 
as the achromatic condenser inverts it once and the objective rein verts 
it (no projection ocular being used). 

If the ordinary hard carbons, without soft core, are used the crater 
shifts its position and thus gives a wavering light. The soft cored 
carbons give a very steady light as the crater remains constant in 
position. 

Sometimes a small hard carbon is used for the lower or negative 
side and a. large soft cored carbon for the upper or positive pole. This 
works admirably. The sizes used by Ayrton were for the positive car- 
bon 13 mm. diameter ; for the negative 11 mm. 

§ 407. Rapidity of Wear in the Carbons. — If one employs two 
carbons equal in size and composition the positive carbon wears away 
twice as fast as the lower one, therefore one will find the feed 
mechanism in many lamps moves the upper carbon down twice as 
fast as the lower carbon moves up. This keeps them centered. 
If one uses a large carbon above and a small one below, and if 
the sizes of upper and lower carbon are properly selected, the two car- 
bons wear away equally in length and the feed mechanism of the lan- 
tern should move the upper carbon down and the lower one up at the 
same rate to insure constant centering. 



254 PROJECTION MICROSCOPE \_CH.IX 

The rapidity of wear in the carbons, irrespective of their position, 
depends upon the amount of the current. It is uneconomical to use 
more current than necessary, both on account of the cost of the elec- 
tricity and the wear of the carbons. It is inconvenient to change the 
carbons too frequently. It is certainly inconvenient to be compelled 
to insert new ones during a demonstration. 

§ 408. Size of Carbons and Amount of Current. — The size 
of the carbons must be proportioned to the amount of current used. 
For an amperage not exceeding 15, a soft cored carbon of 12 mm.(j4 
inch) will answer, but if 20 amperes of current are used then the car- 
bon should be 16 mm. (or f inch) in diameter. If one uses too small 
a carbon for the current, the carbon partly burns, and really less avail- 
able light is produced for the projection.* 

§ 409. Amount of Current for Micro-Projection. — For a lec- 
ture room holding 200 and a screen distance of 8 meters (26 feet), one 
can demonstrate almost any suitable preparation with a current of 10 
to 12 amperes, the voltage being no. This serves for objectives as 
high as 3 mm. when no ocular is used. According to Behrens (Zeit. 
wiss. Mikr. , 1898, pp. 7-23) one cannot make available more than 20 
amperes for any micro-projection. The makers of micro-projection 
apparatus almost invariably make a 20 ampere lamp the limit. 

For getting good results it is vastly more important to have all 
the parts of the apparatus centered and the carbons in the proper rela- 
tive position than to use a powerful current. The light cannot serve 
for projection unless it is properly used (§ 406). 

One will be surprised to see how excellent the results are with an 
amperage of 10 or 12 when one makes the most of the light. With 
some preparations one needs more light, and must increase the amper- 
age. Remember that the skill of the operator is of equal account with 
the amperage. Do not expect the lantern to furnish brains as well as 
light. 

§ 410. Lamp Condenser. — This is a large condenser next the 
radiant and it serves to collect the light emitted from the crater and 



*For the experiments made in preparing this chapter, and for practical use 
during the last two years, the carbons most employed are designated : "High 
grade Electra, Nurnberg carbons, soft cored, 12X190 mm. Other forms were 
also used in the experiments, sometimes the upper carbon was soft cored and the 
lower one solid. With study and careful experiment one can get good results with 
a variety of currents and carbons. The beginner is advised, however to, start with 
the carbons recommended and furnished by the makers of the arc lamp which he 
is to employ. 






CH. IX] 



PROJECT/ON MICROSCOPE 



255 



condense it either upon the object for lantern slides and low objectives, 
or it narrows the light into a cone of the proper size for further con- 
centration by the achromatic condenser (§ 412). One of the most 
used, and also one of the best condensers for the arc lamp is composed 
of two plano-convex lenses with their convex sides facing each other. 
There is then one plane face next the radiant and one toward the mi- 
croscope. The lens next the radiant is somewhat smaller than the 
other. Both are loosely mounted to allow for expansion and the cell 
in which the} 7 are mounted should be freely ventilated. This con- 
denser should be adjustable back and forth and up and down. 




Fig. 206. 

Fig. 206. Arrangement and Centering of the Radiant {Leiss). 

In {/) The radiant, i. e., the crater {Fig. 205) is too far to the right ; 

(2) The a'ater is too far to the left ; 

( 3 ) The crater is too high ; 
{4) The crater is too low ; 

( 5) The crater is too far from the lamp condenser ; 

(6-7) The crater is too near the condenser. 

(8 ) The crater is in the correct position . 

As pointed out in the explanation of Fig. 205, there may be a slight central 
shadow with soft cored carbons when the lamp and condenser are in the best relative 
position. 

§411. "Water Bath for Removing Heat. — This is a cell of 
some kind with plane glass faces. It should be approximately of a 
size to allow the light to pass through a stratum of water 50 mm. 
thick. Even this will allow something over 2% of the heat to pass. 
It is a great advantage to have cool or cold water circulate through this 
constantly. 



256 



PROJECTION MICROSCOPE 



\_CH. IX 



The water bath should be free from the condenser mounting so 
that it may not be heated by conduction (see § 413 for special cooler). 







Fig. 207. 

Fig. 207. Leitz Large Micro-Projection Apparatus {Leitz Catalog). 
In this figure the apparatus is in position for projection with a projection 
ocular. As here shown it consists of : — 

(/) An arc lamp designed for a current up to 20 amperes ; 

(2) A lamp condenser of three lenses, the two inner ones being adjustable ; 

(3 ) A large water bath for removing the heat ; 

(4) Bellows. At one end is a space for the insertion of ordinary lantern 
slides {see the next figure) and at the other end are three condensers of different' 

foci on a revolving nose-piece ; 

(5) Objective stage with a special cooler (Zoih's % 413), and a special slide 
carrier serving as a kind of mechanical stage. 

(6) An objective carrier. This contains a triple, revolving nose-piece at the 
end of the large tube. This tube contains an iris diaphragm and receives an 
adapter for the use of projection oculars. For low objectives and when the projec- 
tion ocular is not to be used, this adapter is removed. Fine and coarse adjustments 
are present for focusing . 

Each part is mdependent and capable of special adjustment. {As shown in the 
next figure, the microscope part may be turned aside, leaving the apparatus for 
ordinary lantern slide projection.) 

§ 412. Special Achromatic Condenser for High Powers. — 
For objectives of 8 mm. and higher there should be a special achro- 
matic condenser next the stage for holding the specimens. This should. 



CH. IX] 



PROJECTION MICROSCOPE 



257 



be adjustable and with centering attachment. For objects which would 
be injured by the heat allowed to pass the large water bath, a special 
cooler is used next the specimen. For such cases the achromatic con- 
denser must be especially constructed or the light would not be focused 
on the object, and one would not get sufficient brilliancy for high 
powers. Special condensers are now made admitting the use of the 
special cooler, and if one has delicate objects which he desires to pro- 
ject, like infusoria, or if he wishes to demonstrate the circulation of 
the blood or living objects he should make provision for it in ordering 
his apparatus. 




Fig. 208. 

Fig. 208. Leitz' Large Projection Apparatus {cut loaned by Wm. Krafft, 
N. V.). 

As here shown the microscope part is turned aside, and the lowest of the three 
condensers (300 mm. equiv. focus) is in place for proj ecting ordinary lantern tides. 

§ 4-!3- Specimen Cooler.— In order to project living objects 
and delicate preparations the heat passing the large water bath must be 
still farther eliminated. This is accomplished by a device of Zoth's 



25§ 



PROJECTION MICROSCOPE 



\_CH. IX 



(Zeit. wiss. Mikr., 1893, p. I 5 2 < J- R- M. S., 1894, P- 112). It consists 
of a metal box with glass covers. Through this box is circulated cold 
water, and the specimen rests directly on the cooler. With this the 
temperature at the focus of the special condenser (§ 412) rarely 
exceeds 27 ° centigrade. 




Fig. 209. 

Fig 209. Spencer Winkel Mechanical Stage. ( Spencer Lens Co. ) This stage is 
convenient for use with the projection microscope as it can be attached to any square 
stage and has a great range of motion. This large range is especially necessary 
for series of organs or embryos. For the projection microscope it would be better 
to have both milled heads for moving the stage on the side as in the next. 

§ 414. Stage for Specimens. — This should be large and prefer- 
ably square or oblong. The central opening should be 50 to 60 mm. in 
diameter, and have a large iris to lessen this diameter at will. The 
specimen stage should be on an independent support and adapted for 
independent motion. This is necessary so that all objects capable of 
projection may be used on the stage and moved far enough from the 



CH. IX] 



PROJECT/ON MICROSCOPE 



259 



microscope for focusing and for the best position of the specimen in 
relation to the other parts of the apparatus (§ 420.) The stage should 
be very rigid. 




Fig. 210. 

Fig. 210. Mechanical Stage of Leitz. {Cut loaned by Wm. Krafft, N. Y.) 
This has the advantage of having both milled heads at the side. It has the disad- 
vantage of not being readily attached to the stage of a projection microscope. 

§ 415. Mechanical Stage. — For projection work this is almost a 
necessity. While one is demonstrating there should be no time and no 
energy wasted in finding the object. Preferably the milled heads of 
the stage should be at the side, and the stage should be easy to remove 
and put back into position. 

It should have sufficient range of motion to enable one to demon- 
strate any section on a slide of serial sections. 

§ 416. Objective Carrier. — This like the stage should be on an 
independent support. There should be both fine and coarse adjust- 
ment. The tube should either be very short, or very large to avoid 
restricting the field. For projection oculars there must be an adapter 
for using them, and the adapter must be long enough to produce the 
proper tube-length (160 mm.). As with the stage the objective car- 
rier should be very substantial. 

§ 417. Objectives to Use in Micro-Projection. — One rarely 
needs an objective lower than 100 mm. focus or higher than 3 mm. 
The majority of the work with a screen distance of 5 to 8 meters will 
be done with objectives of 60-75, 2 5 - 4°> J 6, 8, 6 or 3 mm. focus. For the 



260 



PROJECTION MICROSCOPE 



_ [ CH. IX 



three kept on the nose-piece most constantly a 64, 42 and 6 mm is as- 
good a combination as one can get. Other powers should be available, 
however, and for special specimens and occasions one may employ the 
two mm. oil immersion. For powers above 20 mm., ordinary objectives 
are more satisfactory than most projection objectives. 

§ 4r8. Projection Oculars (Fig. 185). — If one uses the apoch- 
romatic objectives of 16 and 8 mm. focus the projection oculars are 
used with them. They can also be used with wide angle achromatic 
objectives. With them one can get an enormous magnification even 
with the 16 mm. objective. (See the table below, § 421.)- 

These oculars restrict the field greatly and in the writer's exper- 
ience it seemed on the whole more desirable for most objects not to use 
the projection oculars. They cannot be used advantageously with 
objectives higher than 8 or 6 mm. and are most satisfactory with one 
of 16 mm. 

In using the projection ocular with a 16 mm. objective one can 
place the special achromatic condenser next the stage, but if the ocular 
is not used one must either do without a specimen condenser, or take 
one of longer focus, if he has two or three as shown in Fig. 207. 

Fig. 211. Zeiss' Micro-Planar for projection. {Cut 
loaned by Bausch & Lomb Optical Co. ) 

The micro-planars are of 20, jj, jo, j$, and 100 mm. 
equivalent focus . When used for projection the special iris- 
in each should be wide open. These are used without a. 
Fig 2 1 1 projection ocular. 






Fig. 212 A. Fig. 212 B. 

Fig. 212. Leitz objectives of 64 (A), and 42, mm. B). For micro-projection^ 
(Cut loaned by Wm. Krafft, N. Y.) 

These are used without a projection ocular. The iris in the 64 mm. objective- 
should be wide open for micro-projection. 



§4I9- 



Arrangement of the Parts of the Projection Micro- 
scope. — As stated in section 399, all the parts of the apparatus should 






CH. IX] PROJECTION MICROSCOPE 261 

"be adjustable. This is because the arrangement needs to be somewhat 
different for different objectives. 

The guiding principle is that the specimen should be lighted by a 
•converging cone of light, and it should be lighted by the entire cone of 
light traversing the lamp condenser. If one uses a white card it is 
easy to determine the position and size of the cone of light. If it is 
too large for the specimen, either the lamp condenser is too near the 
radiant or the specimen is too close to the lamp condenser. 

If one uses an achromatic condenser the lamp condenser and the 
achromatic condenser should be so arranged that the entire cone of 
light traversing the lamp condenser can enter the achromatic condenser. 
If the cone is too large they are too close together, or the lamp con- 
denser is too near the radiant. If the cone is too small then the lamp 
condenser is too far from the radiant or the achromatic condenser, 
or perhaps both faults are present. One must remember in all his ex- 
periments that a converging cone of light should be used and not a 
diverging one. The specimen must then not. be beyond the focus of 
the lamp condenser. 

If one wishes to make micro-projection a success it will be nec- 
essary to give the apparatus the requisite time and thought. Try to 
understand the conditions of success and continue experimenting until 
you have learned to make it possible for the machine to do its best for 
you. The satisfaction of showing a class real things is sufficient re- 
ward for all the trouble. 

' §420. Screen and Screen Distance. — For a screen nothing is 
so good as a dead-white, smooth wall. A lusterless, white cloth screen 
answers well also. It is an advantage to have this entirely opaque, so 
that none of the light can pass through it. One must remember that 
the light passing through the minute lenses of the objective must be 
spread out over a great space even with low powers, and over a 
much greater with high powers, so that one cannot afford to have any of 
the light lost by transmission through the screen. All are agreed that 
for micro-projection a translucent screen with the projection apparatus 
behind it is not desirable, although for ordinary lantern slide projection 
it answers fairly well. 

The distance of the screen from the microscope depends largely on 
the size of one's audience. The writer has found a distance of eight 
meters (26 feet) good for both low and high power projection. This 
distance answers well for a class of 200 persons. 



262 



PROJECTION MICROSCOPE 



[CH. IX 



For the minute details of a projected specimen it is recommended 
that the audience use opera glasses. These are also useful for the oper- 
ator in focusing the image on the screen. 

TABLE SHOWING THE SIZE OF OBJECT, THE MAGNIFICATION 
AND THE SIZE OF THE SCREEN IMAGE WITH VARIOUS 
OBJECTIVES AND PROJECTION OCULARS 

Distance oj the screen from the stage of the microscope, 8 meters (26 feet +). 
Arc tight, 12 ampere current, for illumination. 



Objective 


Projection 
Ocular 


Achromatic 
Condenser 


Actual Size of 
Field 


Magnification 


Diameter 

of Screen 

Image 


64 mm. 


none 


none 


20 mm. 


125— 


250 cm. 


42 mm. 


none 


none 


10 mm. 


185 


185 cm. 


35 mm. 


none 


none 


9 mm. 


230 


207 cm. 


24 mm. 


none 


none 


7 mm. 


335 


235 cm. 


18 mm. 
" mm. 
" mm. 


none 

X 2 

X 4 


none 
condenser 
condenser 


6 mm. 
1.5 mm. 
1.5 mm. 


440 

860 

1640 


264 cm. 
130 cm. 
245 cm. 


16 mm. 
" mm. 
" mm. 


none 

X 2 

X 4 


none 
condenser 
condenser 


4 mm. 

1.25 mm. 

1 mm. 


53° 
1090 
2040 


200 cm. 
136 cm. 

204 cm. 


8 mm. 

" mm. 
" mm. 


none 
X 2 

X 4 


condenser 
condenser 
condenser 


3+ mm. 
0.60 mm. 
0.60 mm. 


1000 
2250 
4500 


300 cm. 
140 cm. 
270 cm. 


6 mm. 
" mm. 
" mm. 


none 
X 2 
X 4 


condenser 
condenser 
condenser 


2 mm. 
0.50 mm. 
0.50 mm. 


1200 
2550 
5000 


240 cm. 
130 cm. 
250 cm. 


5 mm. 
" mm. 


none 
X 2 


condenser 
condenser 


1.80 mm. 
0.40 mm. 


1750 
3500 


300 cm. 
140 cm. 


4 mm. 


none 


condenser 


1.50 mm. 


2150 


320 cm. 


3 mm. 


none 


condenser 


1 mm. 


2350 


235 cm. 


2 mm. 


none 


condenser 


0.60+mm. 


4000 


300 cm. 



§421. This table shows approximately the size of object which 
each objective will project upon the screen. This was determined ex- 
actly as described in § 50. For the magnification one simply measures 
the distance between two or more lines of the image of the microm- 
eter on the screen, and divides the size of the image by the known 
size of the object ( §155). An)' good stage micrometer will answer. 
It is necessary, however, to use one with coarse lines for the low pow- 
ers (§ 159, 170). 

By comparing the magnification with and without the projection 
oculars, and also comparing the size of object which can be projected 






CH. IX] PROJECTION MICROSCOPE 263 

with and without the oculars one can decide quite accurately the best 
combination to select. On the whole the writer has found it better to 
employ a sufficient variety of objectives and not use the projection 
oculars. It is somewhat easier to obtain a brilliant image without the 
oculars. 

§422. Darkening the Room. — It is impossible to succeed in 
micro- projection unless the room can be made dark, the darker the 
better. It is especially important that the screen should be free from 
all light except that projected upon it in forming the image. 

§423. Enclosing the Projection Apparatus. — It is desirable 
to have the projection apparatus closed as completely as possible to 
avoid diffusing light through the room and thus vitiating the most 
careful darkening of all windows and sky lights. It is also desirable 
to shut in the light from the apparatus, as it dazzles the eyes of the 
operator and of those near it in the audience so that the image on the 
screen cannot be satisfactorily seen. Some forms of apparatus are en- 
closed in a metal box, others have a frame over them upon which is 
spread black cloth like silesia. If this is made fireproof by soaking it 
thoroughly in a solution of alum, borax and sodium tungstate it will 
not readily catch fire. The cloth should not be too thick, otherwise it 
will retain too much heat around the apparatus. 

One should remember the fundamental law of vision, viz, that 
other things being equal, the clearest images are obtained when no light 
reaches the eye except from the object. 

§ 424. Preparations Suitable for Micro-Projection. — As a 
generalization it may be said that any specimen which shows clearly 
and sharply under the microscope with a 16 mm. objective will also 
give an excellent projection image. Details which are not visible 
with the 16 mm. objective are rarely well brought out with sufficient 
clearness on the screen for one or two hundred people to see. 

(A) The stains showing best are those which are very transpar- 
ent, or pure differential stains like hematoxylin. Admirable results 
have been obtained with hematoxylin and eosin, and the various car- 
mines when differentiated. Every method of staining which gives 
either sharply differentiated results or transparent colors produces 
preparations adapted to projection. A weak, or washed out appearance 
under the microscope is sure to be even less satisfactory on the screen. 

(B) The thickness of the sections may vary from \jj. to 40/^. But 
one must remember that thick sections are adapted for low powers 



264 PROJECTION MICROSCOPE \CH. IX 

only, while thin sections, if well stained, may be used with both high 
and low objectives. 

The size of the object which one wishes to project determines the 
objectives to be used. By consulting the table one can get a fair idea 
of the size of object which each objective will satisfactorily project. 
An excellent plan to follow is that for ordinary microscopic study (see 
p. 102), that is, use first a low power to show the object as a whole, then 
a higher one for details. 

(C) For minute objects like white blood corpuscles, etc., it is 
necessary to use a high power and to have a small audience which can 
be close to the screen, or a somewhat larger audience can see well by 
using opera glasses. 

(D) For the circulation of the blood it is necessar}- to eliminate 
the heat rays almost entirely. Nothing has proven so good as the 
second or specimen cooler (§413). The conditions are still more 
favorable if a circulation of cold water is established in the large water 
bath also. This is easily done by the use of two large bottles. The 
cold water can be siphoned or aspirated from an upper one and the 
warm water allowed to flow off into a lower one. For this it is of much 
advantage to have a tube in the bottom of the water bath in which to 
introduce the cold water. The warm water will then flow off through 
a tube in the top. One must remember that perfectly clean water must 
be used for the water bath especially when a circulation is established, 
for opaque particles in the water bath give undesirable shadows in the 
image. 

(B) A practical suggestion is made by Lewis Wright in his book 
on optical projection, and that is to warm the objective before using it 
for showing the circulation of the blood or in any case when a moist 
object is under it. If the objective is cold the vapor from the object 
will be condensed on the objective and make satisf actor)' projection 
impossible. 

§ 425. Masks for Projection Preparations. — The light used for 
projection is so brilliant that it is practicall} 7 impossible to arrange the 
object under the objective with rapidity and certainty unless there is 
some kind of guide. The best one found so far is a mask on the back 
of the slide with an opening for the preparation L to be shown. This 
mask should be made of black paper. One can cut the holes in it with 
scissors or with ticket punches. With the specimens properly masked, 
and the parts of the apparatus lusterless black, as suggested by Dr. 



CH. IX] 



PROJECTION MICROSCOPE 



265 



Coplin, the operator can work with rapidity, certainty and also with 
comfort. (Fig. 213.) 



5_ 
10 

T- 



79 



86 



93 




"1 



Homo 


5 


Slide 


10 


Sec' 8 


79 


20m 


100 


1901 



Fig. 213. Slide of serial sections with a black mask, perforated over the sec- 
tions to be demonstrated with the projection microscope. This mask is put on the 
back of the slide, not on the cover-glass. 

Unless one has a mask something like this the light is so dazzling that it is 
almost impossible to find the proper sections. It is easily removed by placing the 
slide on wet blotting paper. 

§ 426. How to demonstrate with the Micro-Projection Ap- 
paratus. — Microscopical preparations are not so easily used as are lan- 
tern slides. The writer has found that the most successful method is 
for the teacher himself to stand by the apparatus, insert the specimens, 
and find exactty what he wishes his pupils to see. Then to point 
them out a bamboo fish pole with sharp end is used. This should be 
2-3 meters long and if held out in the diverging cone of light leaving 
the microscope, a sharp shadow will be cast upon the image. With 
this pointer one can indicate the part to be demonstrated even more 
satisfactorily than as if he pointed them out directly on the screen. 
While it is not possible to delegate the finding of the specimen to an 
assistant he is of great help in keeping the carbons of the lamp in ex- 
actly the right position. If the light is kept perfect the teacher has 
very little trouble with the rest of the manipulation. 

§427. Cleaning the Glass Surfaces of the Micro-projection 
Apparatus. — Inasmuch as it is so difficult to make the light sufficiently 
brilliant for micro-projection, it is of the greatest importance that all 
glass surfaces be kept as clean as possible. The lenses of the lamp 
condenser should be carefully wiped occasionally ; and the water bath 
should be opened and the plane glass faces thoroughly cleaned. It 
is desirable to soak them in the cleaning mixture for glass. There is 
always a certain amount of deposit on the glass even though distilled 
water is used. Every grade of opacity renders the image on the screen 
less excellent. Cleanliness is one of the most important requirements 
for successful micro-projection. 



266 



PROJECTION MICROSCOPE 



[CH. IX 



Each preparation should be wiped off before it is put in position 
on the stage. Any particles of dust are painfully evident in the pro- 
jected image. 




Fig. 214. Zeiss Epidiascope for Opaque Objects, and for Transparent Objects 
in a Horizontal Position [Zeiss' 1 Special Catalog.) 

As shown in this figure the apparatus is set up for opaque objects. For trans- 
parent objects M 2 [mirror 2) is removed, when the light striking Mi is reflected to 
M 4 and thence up through the object to M 1 and to the screen. 

Commencing at the right : R. Parabolic reflector, which projects the light from 
the crater through ( W ) the water bath, to M 2 the mirror which is at the proper an- 
gle for reflecting the light down upon the opaque object. From the opaque object the 
light is irregularly reflected up through the objective to M 1 . M 1 serves to reflect 
the rays from the objective to the screen. 

V. Ventilator. 3P and M 4 are mirrors for use in reflecting the light through 
horizontal transparent objects. 



CH. IX ] PROJECTION MICROSCOPE 267 

This apparatus is designed to project opaque objects as large as 22 centimeters 
in diameter, at a magnification 0/5 to 10 with a jo ampere current. For a smaller 
object one may magnify as high as 25 diameters. With a 50 ampere current and a 
larger reflector the magnification may be from 14 up to jj diameters. 

PROJECTION OF OPAQUE OBJECTS 

\ 428. Episcope.— For the projection of opaque objects like anatomical prep- 
arations, figures in books, coins or indeed any opaque object an apparatus on the 
principle of the one figured (Fig. 214) is used. That is, a powerful light is thrown 
upon the opaque object and the rays reflected from the object are then projected 
upon the screen by an objective as for a lantern slide. As the objects are mostly 
in a horizontal position the objective points directly upward, and the rays from it 
must be made horizontal by means of a 45 degree mirror or prism. 

This apparatus is very old. Its first name was "aphengescope" or opaque lan- 
tern. Now it is called an episcope, or megascope, and if for both opaque and 
transparent objects (Fig. 214) it is designated as an epidiascope. 

For its satisfactory use exceedingly powerful light must be used. Some opti- 
cians employ two radiants, others but one. In any case currents of 30 to 50 am- 
peres are used. One should wear deeply stained glasses in working with it. 

The apparatus works well with flat objects, and rather brilliant objects, like 
the movements of a watch, etc. It is also more satisfactory for objects of slight 
thickness. For objects like bones, etc., one must focus up and down for the dif- 
ferent levels. 

REFERENCES TO CHAPTER IX 

Lewis Wright, Optical Projection ; Carpenter-Dallinger ; L,eiss, Die optischen 
Instrumente der Firma R. Fuess ; The works on Photo-Micrography ; The latest 
catalogs or special catalogs on projection apparatus issued by the opticians, especial- 
ally Zeiss, Reichert and Leitz. The volumes of the microscopical periodicals for 
the last few years, especially the Journal of the Royal Microscopical Society, and 
the Zeitschrift fiir wissentschaftliche Mikroskopie. 



CHAPTER X 

THE ABBE TEST PLATE AND APERTOMETER ; EQUIVA- 
LENT FOCUS OF OBJECTIVES AND OCULARS ; DRAW- 
INGS FOR PHOTO-ENGRAVING ; WAX MODELS 



\ 429. On the Method of Using Abbe's Test-Plate. — This test-plate is in- 
tended for the examination of objectives with reference to their corrections for 
spherical and chromatic aberration and for estimating the thickness of the cover- 
glass for which the spherical aberration is best corrected. 

"The test-plate consists of a series of cover-glasses ranging in thickness from 
0.09 mm. to 0.24 mm., silvered on the under surface and cemented side by side on 
a slide. The thickness of each is written on the silver film. Groups of parallel 
lines are cut through the film and these are so coarsely ruled that they are easiky 
resolved by the lowest powers, yet from the extreme thinness of the silver they 
form a very delicate test for objectives of even the highest power and widest 
aperture. To examine an objective of large aperture the plates are to be focused 
in succession observing each time the quality of the image in the center of the 
field and the variation produced by using alternately central and very oblique 
illumination. When the objective is perfectly corrected for spheircal aberration 
for the particular thickness of cover-glass under examination, the contour of the 
lines in the center of the field will be perfectly sharp by oblique illumination 
without any nebulous doubling or indistinctness of the minute irregularities of 
the edges. If after exactly adjusting the objective for oblique light, central 
illumination is used no alteration of the adjustment should be necessary to show 
the contours with equal sharpness." 

"If an objective fulfills these conditions with any one of the plates it is free 
from spherical aberration when used with cover-glasses of that thickness ; on the 
other hand if every plate shows nebulous doubling or an indistinct appearance of 
the edges of the silver lines, with oblique illumination, or if the objective requires 
a different adjustment to get equal sharpness with central as with oblique light, 
then the spherical correction is more or less imperfect. ' ' 

"Nebulous doubling with oblique illumination indicates overcorrection of the 
marginal zone, want of the edges without marked nebulosity indicates under- 
correction of this zone ; an alteration of the adjustment for oblique and central 
illumination, that is, a difference of plane between the image in the peripheral 
and central portions of the objective points to an absence of concurrent action of 
the separate zones, which may be due to either an average under or overcorrection 
or to irregularity in the convergence of the ra3 r s. " 

"The test of chromatic correction is based on the character of the color bands, 
which are visible by oblique illumination. With good correction the edges of the 



CH. X] 



TEST PLATE AND APERTOMETER 



269 



silver lines in the center of the field should show but narrow color bands in the 
complementary colors of the secondary spectrum, namely, on one side yellow- 
green to apple-green on the other violet to rose. The more perfect the correction 
of the spherical aberration the clearer this color band appears." 

"To obtain obliquity of illumination extending to the marginal zone of the 
objective and a rapid interchange from oblique to central light Abbe's illuminat- 
ing apparatus is very efficient, as it is only necessary to move the diaphragm in 
use nearer to or further from the axis by the rack and pinion provided for the 
purpose. For the examination of immersion objectives, whose aperture as a 
rule is greater than 180 in air and those homogeneous-immersion objectives, 
which considerably exceed this, it will be necessary to bring the under surface of 
the Test-plate into contact with the upper lens of the illluminator by means of a. 
drop of water, gtycerin or oil." 

"In this case the change from central to oblique light may be easily effected by 
the ordinary concave mirror but with immersion lenses of large aperture it is im- 
possible to reach the marginal zone by this method, and the best effect has to be 
searched for after each alteration of the direction of the mirror." 

"For the the examination of objectives of smaller aperture (less than 4o°-5o°). 
we may obtain all the necessary data for the the estimation of the spherical and 
chromatic corrections by placing the concave mirror so far laterally, that its edge 
is nearly in the line of the optic axis the incident cone of rays then only filling 
one-half of the aperture of the objective. The sharpness of the contours and the 
character of the color bands can be easily estimated. Differences in the thickness 
of the cover-glass within the ordinary limits are scarcely noticeable with such 
objectives." 

"It is of fundamental importance in employing the test as above described to- 
have brilliant illumination and to use an eye-piece of high power." 

"When from practice the eye has learnt to recognize the finer differences in 
the quality of the contour images this method of investigation gives very trust- 
worthy results. Differences in the thickness of cover glasses of 0.01 or 0.02 mm. 
can be recognized with objectives of 2 or 3 mm. focus." 

"With oblique illumination the light must always be thrown perpendicularly 
to the direction of the lines. 




Fig. 215. The Abbe Test Plate. 

"The quality of the image outside the axis is not dependent on spherical and 
chromatic correction in the strict sense of the term. Indistinctness of the con- 
tours towards the borders of the field of view arises as a rule, from unequal mag- 
nification of the different zones of the objective ; color bands in the peripheral 



270 TEST PLATE AND APERTOMETER \CH. X 

portion (with good color correction in the middle) are caused by unequal magnifi- 
cation of the different colored images." 

"Imperfections of this kind, improperly called "curvature of the field," are 
shown to a greater or less extent in the best objectives, where the aperture is con- 
siderable." 



Fig. 216. Abbe Apertometer. 

§430. Determination of the Aperture of Objectives with an Apertometer. — 
Excellent directions for using the Abbe apertometer may be found in the Jour. 
Roy. Micr. Soc, 1878, p. 19, and 1880, p. 20 ; in Dippel, Zimmermann and Czapski. 
The following directions are but slightly modified from Carpenter-Dallinger, pp. 
394-396. The Abbe apertometer involves the same principle as that of Tolles, but 
it is carried out in a simpler manner ; it is shown in Fig. 216. As seen \>y this figure 
it consists of a semi-circular plate of glass. Along the straight edge or chord the 
glass is beveled at 45 , and near this straight edge is a small, perforated circle, the 
perforation being in the center of the circle. To use the apertometer the micro- 
scope is placed in a vertical position, and the perforated circle is put under the mi- 
croscope and accurately focused. The circular edge of the apertometer is turned 
toward a window or plenty of artificial light so that the whole edge is lighted. 
When the objective is carefully focused on the perforated circle the draw-tube is 
removed and in its lower end is inserted the special objective which accompanies 
the apertometer. This objective and the ocular form a low power compound mi- 
croscope, and with it the back lens of the objective, whose aperature is to be meas- 
ured, is observed. The draw-tube is inserted and lowered until the back lens of 
the objective is in focus. "In the image of the back lens will be seen stretched 
across, as it were, the image of the circular part of the apertometer. It will ap- 
pear as a bright band, because the light which enters normally at the surface is re- 
flected by the beveled part of the chord in a vertical direction so that in reality a 
fan of 180 in air is formed. There are two sliding screens seen on either side of 
the apertometer ; they slide on the vertical circular portion of the instrument. 
The images of these screens can be seen in the image of the bright band. These 
screens should now be moved so that their edges just touch the periphery of the back 
lens. They act, as it were, as a diaphragm to cut the fan and reduce it, so that its 
angle just equals the aperature'of the objective and no more." "This angle is 
now determined by the arc of glass between the screens ; thus we get an angle in 
glass the exact equivalent of the aperature of the objective. As the numerical ap- 
ertures of these arcs are engraved on the apertometer they can be read off by inspec- 
tion. Nevertheless a difficulty is experienced, from the fact that it is not easy to 



CH.X~\ TEST PLATE AND APERTOMETER 271 

determine the exact point at which the edge of the screen touches the periphery 
of the back lens, or as we prefer to designate it, the limit of aperture, for curious 
as the expression may appear we have found at times that the back lens of an ob- 
jective is larger than the aperture of the objective requires. In that case the 
edges of the screen refuse to touch the periphery." 

In determining the aperture of homogeneous immersion objectives the proper 
immersion fluid should be used as in ordinary observation. So, also, with glycerin 
or water immersion objectives. 

\ 431. Testing Homogeneous Immersion Liquid. — In order that one shall 
realize the full benefit of the homogeneous immersion principle it is necessary 
that the homogeneous immersion liquid shall be truly homogeneous. In order 
that the ordinary worker may be able to test the liquid used by him, Professor 
Hamilton L. Smith devised a tester composed of a slip of glass in which was 
ground accurately a small concavity and another perfectly plain slip to act as 
cover. (See Proc. Amer. Micr. Soc. ; 1885, p. 83). It will be readily seen that 
this concavity, if filled with air or any liquid of less refractive index than glass, 
will act as a concave or dispersing lens. If filled with a liquid of greater refractive 
index than glass, the concavity would act like a convex lens, but if filled with a 
liquid of the same refractive index as glass, that is, liquid optically homogeneous 
with glass, then there would be no effect whatever. 

In using this tester the liquid is placed in the concavity and the cover put on. 
This is best applied by sliding it over the glass with the concavity. A small 
amount of the liquid will run between the two slips, making optical contact on 
both surfaces. One should be careful not to include air bubbles in the concavity. 
The surfaces of the glass are carefully wiped so that the image will not be ob- 
scured. An adapter with society screw is put on the microscope and the objective 
is attached to its lower end. In this adapater a slot is cut out of the right width 
and depth to receive the tester which is just above the objective. As object it 
is well to employ a stage micrometer and to measure carefully the diameter of the 
field without the tester, then with the tester far enough inserted to permit of the 
passage of rays through the glass but not through the concavity, and finally the 
concavity is brought directly over the back lens of the objective. This can be 
easily determined by removing the ocular and looking down the tube. 

Following Professor Smith's directions it is a good plan to mark in some way 
the exact position of the tube of the microscope when the micrometer is in focus 
without the tester, then with the tester pushed fc in just far enough to allow the light 
to pass through the plane glass and finally when the light traverses the concavity. 
The size of the field should be noted also in the three conditions ( \ 50-52. ) 

It will be seen by glancing at the following table that whenever the liquid in the 
tester is of lower index than glass, that the concavity with the liquid acts as a 
concave lens, or in other words like an amplifier (p. 109), and the field is smaller 
than when no tester is used. It will also be seen that as the liquid in the concav- 
ity approaches the glass in refractive index that the field approaches the size 
when no tester is present. It is also plainly shown by the table that the greater 
the difference in refractive index of the substance in the concavity and the glass, 
the more must the tube of the microscope be raised to restore the focus. 

If a substance of greater refraction than glass is used in the tester the field 
would be larger, i. e., the magnification less, and one would have to turn the tube 
down instead of up to restore the focus. 



272 



TEST PLATE AND APERTOMETER 



CH. X] 



The table given below indicates the points with a tester prepared by the Gund- 
lach Optical Co., and used with a 16 mm. apochromatic objective of Zeiss, X4 
compensation ocular, achromatic condenser, 1.00 N. A. (Fig. 41) : 



Tester aud Liquid in the Concavity 



end, 



No tester used 

Whole thickness of the tester at one 

not over the cavity 

Tester with water 

Tester with 95 % alcohol 

Tester with kerosene 

Tester with Gundlach Opt. Co's hom. liquid 

Bausch & Lomb Opt. Co.'s hom. liquid . 

Leitz' hom. liquid 

Zeiss' hom. liquid 



Size of the 
Field 



1.825 mm. __ 

1.85 mm. 

1.075 mm. 

1. 15 mm. 

1.4 mm. 

1.825 mm. 

1.825 mm. __ 

1.825 mm. 

1.825 mm. __ 



Elevation of the Tube 

necessary to 

Restore the Focus 



Standard position. 

No change of focus. 
Tube raised 2,% mm. 

3 mm. 

2 mm. 



\ 432. Equivalent Focus of Objectives and Oculars. — To work out in proper 
mathematical form or to ascertain experimentally the equivalent foci of these 
complex parts with real accuracy would require an amount of knowledge and of 
apparatus possessed only by an optician or a physicist. The work may be done, 
however, with sufficient accuracy to supply most of the needs of the working 
microscopist. The optical law on which the following is based is : — "The size of 
object and image varies directly as their distance from the center of the lens.' 1 '' 

By referring to Figs. 14, 16, 21, it will be seen that this law holds good. 
When one considers compound lens-systems the problem becomes involved, as the 
centre of the lens systems is not easily ascertainable hence it is not attempted, 
and only an approximately accurate result is sought. 

\ 433- Determination of Equivalent Focus of Objectives. — Look into the 
upper end of the objective and locate the position of the back lens. Indicate the 
level. in some way outside of the objective. This is not the center of the object- 
ive but serves as an arbitrary approximation. Screw the objective into the tube 
of the microscope. If a Huygenian ocular is used with the ocular micrometer, 
screw off the field lens and use the eye-lens only. If a positive ocular is used 
no change need be made. Pull out the draw-tube until the distance between the 
ocular micrometer and the back lens is 250 millimeters. Use a stage micrometer as- 
object and focus carefully. Make the lines of the two micrometers parallel 
(Fig. 108). Note the number of spaces on the ocular micrometer required to 
measure one or more spaces on the stage micrometer. Suppose the two microm- 
eters are ruled in y 1 ^ mm. and that it required 10 spaces on the ocular micrometer 
to enclose 2 spaces on the stage micrometer, evidently then 5 spaces would cover 
one. The image, A I B I Fig 21 in this case is five times as long as the object, A,B. — 
Now if the size of object and image are directly as their distance from the lens it 
follows that as the size of object is known ( t 2 q mm. ), that of the image directly 
measured (\% mm.), the distance from the lens to the image also determined in 
the beginning, there remains to be found the distance between the objective and 
the object, which will represent approximately the equivalent focus. The general 
formula is, Object, O: Image, I : :equivalent focus, F: 250. Supplying the known 



CJT.X] TEST PLATE AND APERTOMETER 273 

values, 0= t 2 q, \=\% then T ^ m. : 1 mm. :; F : 250 whence F=5o mm. That is, the 
equivalent focus is approximately 50 millimeters. 

§434. Determination of Initial or Independent Magnification of the Objec- 
tive. — The initial magnification means simply the magnification of the real image 
(-VB 1 , Fig. 21) unaffected by the ocular. It may be determined experimentally 
exactly as described in \ 433. For example, the image of the object ( T 2 „ mm. ) 
measured by the ocular micrometer, at a distance of 250 mm. is \% mm., i. e., it is 
five times magnified, hence the initial magnification of the 50 mm. objective is 
approximately five. 

Knowing the equivalent focus of an objective, one can determine its initial 
magnification by dividing 250 mm. by the equivalent focus in millimeters. Thus 
the initial magnification of a 5 mm. objective is -f a — 50 ; of a 3 mm., -2-f- 2 - —83.3 ; 
of a 2 mm., - 2 -§- a = 125, etc. 

I 435. Determining the Equivalent Focus of an Ocular. — If one knows the 
initial magnification of the objective (§434) the approximate equivalent focus of 
the ocular can be determined as follows : 

The field lens must not be removed in this case. The distance between the 
position of the real image, a position indicated in the ocular by a diaphragm, and 
the back lens of the objective should be made 250 mm., as described in \ 433, 434, 
then by the aid of Wollaston's camera lucida the magnification of the whole mi- 
croscope is obtained, as described in \ 160. As the initial power of the objective 
is known, the power of the whole microscope must be due to that initial power 
multiplied by the power of the ocular, the ocular acting like a simple microscope 
to magnify the real image (Fig. 21). 

Suppose one has a 50 mm. objective, its initial power will be approximately 5. 
If with this objective and an ocular of unknown equivalent focus the magnification 
of the whole microscope is 50, then the real image or initial power of the objective 
must have been multiplied 10 fold. Now if the ocular multiplies the real image 
10 fold it has the same multiplying power as a simple lens of 25 mm. focus, for, 
using the same formula as before : = 5:1 = 50 : : F: 250 whence F = 25. The 
matter as stated above is really very much more complex than this, but this gives 
an approximation. 

For a discussion of the equivalent focus of compound lens-systems, see 
modern works on physics ; see also C. R. Cross, on the Focal Length of Micro- 
scopic Objectives, Franklin Institute Jour., 1870, pp. 401-402 ; Monthly Micr. 
Jour., 1870, pp. 149-159 J. J. Woodward on the Nomenclature of Achromatic 
Objectives, Amer. Jour. Science, 1872, pp. 406-414; Monthly Micr. Jour. , 1872, pp. 
66-74. W. S. Franklin, method for determining focal lengths of microscope 
lenses. Physical Review, Vol. I, 1893, p. 142. See pp. 1119-1131 of Carpenter- 
Dallinger for mathematical formulae ; also Daniell, Physics for medical students ; 
Czapski, Theorie der optischen Instrumente ; Dippell, Nageli und Schwendener, 
Zimmermann. E. M. Nelson, J. R. M. S. 1898, p. 362, 1900, pp. 162-169. J our - 
Quekett Micr. Club, vol. V. pp. 456, 462. 

\ 436. Drawings for Photo-Engraving. — The inexpensive processes of repro- 
ducing drawings bring within the reach of every writer upon scientific subjects 
the possibility of presenting to the eye by diagrams and drawings the facts dis- 
cussed in the text. Though artistic ability is necessary for perfect representation 
of an object, neatness and care will enable anyone to make a simple illustrative draw- 
ing, from which an exact copy can be obtained and a plate prepared for printing. 



274 APPARATUS FOR SECTIONING [_CH. X 

A careful stud}' of the cuts or plates used to illustrate the same class of facts 
as one wishes to show will enable one to produce similar effects. Out- 
lines which are transferred to the drawing paper may be obtained by the camera 
lucida or from a photograph. The drawing should be made so that it can be 
reduced anywhere from one-eighth to one-half. For ordinary photo-engraving 
for such line drawings as are used to illustrate this book, use perfectly black 
carbon ink. A shaded or wash drawing can be reproduced by the half-tone 
process, also photographs as is illustrated by figures 190-191. A crayon drawing 
on stipple paper with shadows re-enforced by ink lines and high lights scratched out 
with a sharp knife give admirable results for anatomical figures by the half-tone 
process. (See for example the work of Max Broedelin Contributions to the Science 
of Medicine, (Welch Book) Baltimore, 1900). 

For photo-engravings of line work the letters, figures or words used to desig- 
nate the different parts can be put on the drawing by pasting letters, etc., of the 
proper size in the right position. In preparing the block the photo-engraver 
eliminates all shadows and the letters look as if printed on the drawings. 

\ 437. Wax Models. — Large wax models of the objects which one studies 
under the microscope are helpful both to the teacher and to the investigator. 
These models are becoming more and more appreciated for embryologic and 
morphologic investigations, for, as one can readily appreciate, the effort to produce 
a representation of the embryo or organ in three dimensions helps to overcome 
difficulties which are almost insurmountable if studied in the sections alone. 

They are made from wax plates, the principle involved being that the diame- 
ter of the drawing on the wax plate is as much greater than the object as the wax 
plate is thicker than the section. 

The wax plate is cut with a sharp instrument, following the outlines of the 
object which has been traced upon it by the aid of a camera lucida or the projec- 
tion microscope. The sections are piled together, some line or lines obtained 
from a drawing or photograph of the spceimen before it was imbedded and sec- 
tioned being used as a guide by which the correct form of the pile of sections can 
be tested. Finally the whole is welded into one by the use of hot wax or a hot 
instrument. Models which illustrate complex internal structures are difficult to 
prepare, but numerous devices will occur to the worker as the representation of 
blood vessels and nerves by strings or wires. A large model will need much sup- 
port which can be given by wire gauze, wires, pins or paper according to the 
special needs. 

A practical method for wax modeling was first published by G. Born, Arch. f. 
Mikr. Anat., Bd. xxii, 1883, p. 5S4. The most detailed statements of improve- 
ments of the method have been published by Born (B6hm u. Oppel) 1900, and by 
Dr. F. P. Mall and his assistants. See contributions to the Science of Medicine, 
pp. 926-1045. Proceedings of the Amer. Assoc. Anatomists, 1901, 14th session 
(1900) p. 193. 

\ 43S. Some Apparatus for Imbedding and Sectioning. — As a supplement to 
Chapter VIII, the following figures of imbedding and sectioning apparatus are 
appended. It will be noticed that the microtomes are complex and consequently 
expensive. One is figured in which the knife is moved by the hands of the oper- 
ator (Fig. 217). This form of instrument is excellent, and with it one can do 
all kinds of work, both with collodion and paraffin. One cannot work so rapidly 



CH. X] 



APPARATUS FOR SECTIONING 



275 



nor with the same precision. For much of the work one may section free-hand, 
without a microtome. Indeed the great basis of histological and embryological 
knowledge was gained by studying free-hand sections and dissections. At the 
present time there is a strong reaction against the exclusive study of sections, 
and a tendency to combine with the serial sections dissections such as the older 
anatomists and embryologists made and gained so much from. 




Fig. 217. A Microtome for 
all kinds of sectioning ; the 
knife is guided by the top of the 
microtome, but moved by the 
hands of the operator (Bausch & 
Lomb Optical Co.) 




Fig. 218. The Minot microtome for ribbon sections as made by Bausch and 
Lomb Optical Co. It is arranged for sections from iju to 2511 and any intermedi- 
ate thickness. 



276 



APPARATUS FOR SECTIONING 

EZZES 



[ CH. X 




Fig. 219. The Minot microtome for ribbon sections as made by the Franklin 
Laboratory Supply Co., Boston. This is to be made for 21.1, 6u, ion, 1411, son, and. 
jou sections. 




A 



Fig. 220. 





Fig. 221. A. 



CH. X ] 



APPARATUS FOR SECTIONING 



277 




Fig. 22 r. i>\ 




Fig. 221. 




A B 

Fig. 222. 

Fig. 220-222. A paraffin holder clamp and a razor siipportfor the Minot Mi- 
crotome. ( Trans. Amer. Micr. Soc, igoi). 

Fig. 220. Clamp for the paraffin block holder. In A it is shown in section, in 
a side view. With this clamp one can use stove bolts as well as the expensive par- 
affin holders furnished with the instrument. A laboratory can have as many par- 
affin block holders as necessary without undue expense. 

Fig. 221. Razor Support and Razor. 

(A) Support with heavy base and vertical piece. The base should be capable 
of moving endwise one or two centimeters to bring Vie opening in the vertical part 
opposite the paraffin block. 

(B) Front piece to the razor {see Fig. 222 A ). 

( C) Razor with straight back and edge. By moving this back and forth on 
the support nearly the entire cutting edge can be utilized. 

Fig. 222. The knife support of the microtome with the razor support and 
razor in position. 

{A) Front view ; (B) Back view. In the inclination of the knife toward the 
paraffin block is shown. 



278 



APPARATUS FOR SECTIONING 



\CH. X 




Fig. 223. Sliding microtome adapted especially for collodion sectioning. ( The 
Bausch & Lomb Optical Co.). 



CH. X] 



APPARA TUS FOR SECTIONING 



279 




A 




Fig. 224 Paraffin dish for infiltrating in the Lillie oven. It is made of cop- 
per and as shown has a handle for ease in transference. A the whole dish, B the 
dish in section. (Jour. Appl. Micr. iSgg, p. 266). 




Fig. 225. The Lillie compartment, paraffin oven for infiltrating tissues with 
paraffin. Various sizes of this are made (<?, 16 and 24 compartments). Except for 
the largest laporatories the one with 16 compartments and trays will be found of suf- 
ficient capacity. 1 ' Bausch & Lomb Optical Co. ). 



28o 



APPARATUS FOR SECTIONING 



\CH. X 




Fig. 226. Circulation board, especially for Necturus. This is prepared from 
a board about 8 x 20 centimeters. Near one edge it has a hole for a perforated cork. 
On the top of the cork is cemented a thick cover-glass with shellac or rubber cement. 
The cork can be raised or lowered in the board. The gills of Necturus or the 
web of 'a frog' 's foot can be spread out on glass over the cork. (four. Appl. Micr., 
1898 , p. 131. 



Fig. 227. Copper can with screw top for collecting 
embryologic material and small aquatic animals. It was 
especially designed for collecting with a bicycle (four. 
Appl. Micr., 1898, p. 131). 





Fig. 228. Egg pipette. This is made by putting a 
short piece of soft rubber tubing over the end of a glass 
pipette with rubber bulb. With this one can handle the 
eggs both fresh and hardened without any degree of in- 
jury, (four. Appl. Micr. 1898, p. 129). 




Fig. 229. Washing apparatus for tissues fixed in osmic and chromium mix- 
tures. As shown i?i the figure the apparatus is connected with the water pipe by a 
small side cock. It is composed of a double vessel, the inner one being made of per- 
forated brass. There are special perforated dishes to insert in the little compart- 
ments. This apparatus is convenient for washing cover-glasses, for the washing 
out for iron hematoxylin, etc. The deeper box at the right answers for the slide 
baskets or holders ( Fig. 139) . 



CH. X~\ 



APPARATUS FOR SECTIONING 




Fig. 229 A. — Same as the preceding with the inner, perforated box on edge. 



Name of Article 



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Fig. 230. An inventory card for the property in a department. The cards are 
the standard size for Libraries, {four. Appl. Micr. , 1898, p. 124). 



BIBLIOGRAPHY 



The books and periodicals named below in alphabetical order pertain wholly or in part to the 
microscope or microscopical methods. They are referred to in the text by recognizable abbrevi- 
ations. 

For current microscopical and histological literature, the Journal of the Royal Microscopical 
Society, the Zoologischer Anzeiger, and the Zeitschrift fur wissenschaftliche Mikroskopie, Ana- 
tomischer Anzeiger, Biologisches Centralblatt and Physiologisches Centralblatt, the Journal of 
Applied Microscopy and laboratory methods and the smaller microscopical journals taken to- 
gether furnish nearly a complete record. 

References to books and papers published in the past may be found in the periodicals just 
named, in the Index Catalog of the Surgeon General's library ; in the Royal Society's Catalog of 
Scientific Papers, and in the bibliographical references given in special papers. A full list of peri- 
odicals may also be found in Vol. XVI of the Index Catalog. 

BOOKS. 

Adams, G. — Micrographia illustrata, or the microscope explained, etc. Illustrated. 4th edi- 
tion, Eondon, 1771. Also Essays, 1787. 

Angstrom. — Recherches sur le spectre solaire, spectre normal du soleil. Upsala, 1868. 

Anthony, Wm. A., and Brackett, C. F. — Elementary text-book of physics. 7th ed. Pp. 527, 165 
Fig. New York, 1891. 

Barker - — Physics. Advanced course. Pp. 902, 380 Fig. New York, 1892. 

Bausch, E. — Manipulation of the Microscope. A manual for the work table and a text-book 
for the beginners in the use of the microscope, illustrated. New Edition, 1901. Rochester, N. Y. 

Beale, E- S. — How to work with the microscope. 5th ed. Pp. 518, illustrated. London, 1880. 
Structure and methods. 

Beauregard H., et Galippe, V. — Guide de l'elfeve et du praticien pour les travaux pratiques de 
micographie, comprenant la technique et les applications du microscope a, l'histologie vegetale, 
a la physiologie, a la clinique, a la hygifene et a la medicine legale. Pp. 904, 570 Fig. Paris, 1880. 

Behrens, H — Mikrochemische Technik, 2d ed. Pp. 68. Hamburg, 1900. 

Behrens, T. H. — Anleitung zur microchemischen Analyse der wichtigsten organischen Ver- 
bindungen. Hamburg, 1S95-1897. 

Behrens, T. H. — Transl, by J. W. Judd. A manual of microchemical analysis with an intro- 
ductory chapter by J. W. Judd. L,ondon, 1894. 

Behrens, W. — The microscope in botany. A guide for the microscopical investigation of 
vegetable substances. Translated and edited bj- Hervey and Ward. Pp. 466, illustrated. Boston, 
1885. 

Behrens, W. — Tabellen zum Gebrauch, bei mikroskopischen Arbeiten. 3d edition. Pp. 237. 
Braunschweig, 1S9S. 

Behrens, W., Kossel, A., und Schiefferdecker, P.— Das Mikroscop und die Methoden der 
mikroskopischen Uuntersuchung. Pp. 315, 193 Fig. Braunschweig, 1889 +. 

Boehm, A. A. und von Davidoff. M. — A text-book of Histology, including microscopic tech- 
nique, from the 2d German edition. Translated by H. H. Cushing and edited b3' G. C. Huber. 
Pp. 501, illustrated. Philadelphia and Eondon, 1900. 

Boehm, A. und Oppel, A. — Taschenbuch der mikroskopischen Technik, 4th ed. with direc- 
tions by Born for making wax models. Pp. 240. Miinchen, 1900. 

Brewster, Sir David. — A treatise on the mikroscope. From the 7th ed. of the Encyc. Brit., 
with additions. Illustrated, 1837. 

Brewster, Sir David. — A treatise on optics. Illustrated. New edition. Eondon, 1853. 

Browning, J. — How to work with the micro-spectroscope. Eondon, 1S94. 



BIBLIOGRAPHY 283 

Bousfield, E. C. — Guide to photo-micrography. 2d ed. Illustrated. London, 1892. 

Carnoy, J. B. Le Chanoine. — La Biologie Cellulaire ; Etude comparee de la cellule dans les 
deux regnes. Illustrated (incomplete). Paris, 1S84. Structure and methods. 

Carpenter, W. B. — The microscope and its revelations. 6th ed. Pp. SS2, illustrated. London, 
and Philadelphia, 1881. Methods and structure. 

Carpenter-Dallinger. — The microscope and its revelations, by the the late William B. Carpen- 
ter. Sth edition, in which the 1st seven and the 23d chapters have been entirely re-written, and 
the text throughout reconstructed, enlaiged and revised by the Rev. W. H. Dallinger. 22 plates 
and nearly 900 wood engravings. Pp. 11S1. London and Philadelphia, 1901. 

Chambeilain, C. J. — Methods in plant histology, Illustrated. Chicago, 1901. 

Clark, C. H. — Practical methods in microscopy. Illustrated. Boston, 1894. 

Cooke, M. C. — One thousand objects for the microscope. Pp. 123. London, no date. 500 fig- 
ures and brief descriptions of pretty objects for the microscope. 

Crookshank, E. M. — Photography of bacteria. London and New York, 1887. 

Cross and Cole. — Modern microscopy for beginners. Part I. The microscope and instruc- 
tions for its use. Part II. Microscopic objects, how prepared and mounted. Illustrated. 2d 
edition, London, 1895. 

Czapski, Dr. Siegfried. — Theorie der optischen Instrumente nach Abbe. Illustrated. Bres- 
lau, 1S93. 

Dana, J. D. — A system of mineralogy. Illustrated. 6th ed. New York, 1S92. 

Daniell, A. — A text-book of the principles of physics. Illustrated. 3d ed. London, 1895. 

Daniell, A. — Phj^sics for students of medicine. Illustrated. London and New York, 1896. 

Davis, G. — Practical microscopy. 3d ed. Illustrated. London, 1895. 

Dippel, L- — Das Mikroskop und seine Anwendung. Illustrated. Braunschweig, 1898. 

Dodge, Charles Wright. — Introduction to elementary practical biology ; a laboratory guide for 
high school and college students. New York, 1894. 

Eberth, C. J.— Friedlander's mikroskopische Technik zum gebiauche bei medicinischen und 
pathologische-anatomischen Untersuchungen. Pp. 359, illustrated. Berlin, 1900. 

Ebner, V. V. — Untersuchungen tiber die Ursachen der Anisotropic organischer Substanzen. 
Leipzig, 1882. Large number of references. 

Ellenberger, W T . — Handbuch der vergleichenden Histologic und Physiologie der Haussauge- 
thiere. 2d edition. Berlin, 1901 + . 

Fol, H. — Lehrbuch der vergleichenden mikroskopischen Anatomie, mit Einschluss der ver- 
gleichenden Histologic und Histogenie. Illustrated (incomplete). Leipzig, 1884. Methods and 
structure. * 

Foster, Frank P. — An illustrated encyclopaedic medical dictionary, being a dictionary of the 
technical terms used by writers on medicine and the collateral sciences in the Latin, English, 
French and German languages. Illustrated, four quarto volumes. 1888-1893. 

Fraenkel und Pfeiffer. — Atlas der Bacterien-Kunde. Berlin, 1889 + . 

Francotte, P. — Manuel de technique microscopique. Pp. 433, no Fig. Brussels, 1886. 

Francotte, P. — Microphotographie appliquee S, l'histologie, l'anatomie comparee et l'embryolo- 
gie. Brussels, 1886. 

Fre\-, H. — The microscope and microscopical technology. Translated and edited by G. R. 
Cutter. Pp. 624, illustrated. New York, 1880. Methods and structure. 

Gait, H. — The microscopy of the starches illustrated by photomicrographs. London, 1900. 

Gamgee, A. — A text-book of the physiological chemistry of the animal bod}'. Part I, pp. 487, 
63 Fig. London and New York, 1S80. Part, II, 1893. 

Gebhardt, W. — Die mikrophotographische Aufnahme gefarbter Praparate. Illustrated. 
Mtlnchen, 1899. 

Giltay, Dr. E. — Sieben Objecte unter dem Mikroskop. Einf lihrung in die Grundlehren der 
Mikroskopie. Leiden, 1893. This is also published in the Holland (Dutch) and French language. 

Goodale, G. L. Physiological botany. Pp. 499 + 36, illustrated. New York, 1885. Structure 
and methods. 



284 BIBLIOGRAPHY 

Griffith and Henfrey. — The Micrographic Dictionary : a guide to the examination and investi- 
gation of the structure and nature of microscopic objects. Fourth edition, by Griffith, assisted by 
Berkeley and Jones. London, 1883. 

Gould, G. M. — The illustrated dictionary of medicine, biology and allied sciences. Illustrated, 
5th ed., Philadelphia, 1900. This is recognized as the best single volume medical dictionary. It is 
especially satisfactory for the worker with the microscope. 

Hager, H. und Mez. C. — Das mikroskop und Seine Anwendung. 8th ed., revised and en- 
larged. Pp. 335. Illustrated. Berlin, 1899. 

Halliburton, W. D. — A text-book of chemical physiology and pathology. Pp. 874, 104 illus- 
trated. London and New York, 1891. 

Hanausek, T. F. — Lehrbuch der mikroskopischen Technik. Illustrated. Stuttgart, 1900. 

Harker, H. — Petrology for students ; an introduction to the study of rocks under the micro- 
scope. Cambridge, 1895. 

Harting, P. — Theorie and algemeine Beschreibung des Mikroscopes. 2d ed. 3 vols. Braun- 
schweig, 1866. 

Herzfeld, T, J. — The technical testing of yarns and textile fabrics with reference to official 
specifications. Translated by C. Salter. Illustrated. London, 1898. 

Hogg, J. — The microscope, its histoiy, construction and application. 15th edition, illustrated. 
Pp. 704. London and New York, 1898. Much attention paid to to the polariscope. 

Huber, G. C. — Laboratory work in histology, 3d revised and enlarged edition. Pp. 204, illus- 
trated. Ann Arbor, 1900. 

James, F. L-, — Elementarj' microscopical technology. Part I, the technical history of a slide 
from the crude material to the finished mount. Pp. 107, illustrated. St. Louis, 18S7. 

Jeserich, P. — Die mikrophotographie auf Bromsilbergelatine bei nattirlichem und Kiinstli- 
chem Lichte. Figs, and Plates. Pp. 245. Berlin, 18SS. 

Jtiptner, Hanns Freiherr von. — Grtindzuge der Siderologie, Erster Teil, Leipzig, 1900. Full 
discussion of theory of solution, chemidal constitution, micrography, etc., of iron alloys and 
slags. Illustrated. Bibliography very complete. Part II not yet issued. "Will deal with relation 
between chemical constitution, mechanical treatment, microscopic structure," etc. 

Kahlden, C. von. — Technik der histologischen Unterschuug pathologisch — anatomischer 
Praparate. 6th revised and enlarged edition. Pp. 174. Jena, 1900. 

Kaiser, W. — Die Technik des modernen Mikroskopes. 2d edition. Illustrated- Wien, 1901. 

King, J. — The microscopist's companion. A popular manual of practical microscopy. Illus- 
trated. Cincinnati, 1S59. 

Klement et Reuard. — Reactions microchemiques k cristaux et leur application en analyse 
qualitative. Pp. 126, S plates. Bruxelles, 1886. 

Kraus, G. — Zur Kentniss der Chlorophyllfarbstoffe. Stuttgart, 1872. 

Lankaster, E. — Half-hours with the microscope ; a popular guide to the use of the microscope 
as a means of amusement and instruction. 20th ed. Pp. 150. London, 1898. 

Latteux, P. — Manuel de technique microscopique. 3d ed. Paris, 1887. 

Le Conte, Joseph. — Sight — an exposition of the principles of monocular and binocular vision. 
Pp. 275, illustrated. New York, 1SS1. 

Lee, A. B. — The microtomist's vade-mecum. A hand-book of the methods of microscopic 
anatomy. 5th ed. Pp. 532. Philadelphia, 1900. 

Lehmann, C. G.— Physiological chemistry. 2 vols. Pp. 64S+547, illustrated. Philadelphia, 
1855- 

Lehmann, O.— Molekularphysik mit besonderer Beriicksichtigung mikroskopischer Unter- 
suchungen und Anleitung zu solchen, sowie einem Anhang tiber mikroskopische Analyse. 2 vols. 
Illustrated. Leipzig, 188S-1S89. 

Leiss, C. — Die optischen Instrumente der Firma R. Fuess, deren Beschreibung. Justierung 
und Anwendung. Pp. 397, illustrated. Leipzig, 1899. 

Lockyer, J. N. — The spectroscope and its application. Pp. 117, illustrated. London and New 
York, 1S73 



BIBLIOGRAPHY 285 

Luquer, L- Mc. I. — Minerals in Rock Sections. Practical methods of identifying minerals in 
rock sections with the microscooe. 

MKendrick, J. G.— A text-book of physiology. Vol. I, general physiology. Pp. 516, illus- 
trated. New York, 18S8. 

Mace, E. — Les substances alimentaire etudies au microscope surtout au point de vue de leurs 
alterations et de leur falsifications. Illustrated. Paris, 1891. 

Macdonald, J. D. — A guide to the microscopical examination of drinking water. Illustrated. 
London, 1S75. Methods and descriptions. 

MacMunn, C. A. — The spectroscope in medicine. Pp. 325, illustrated. London, 1885. 

Marktanner-Turneretscher, G. — Die Mikro-Photographie als Hilfsmittel naturwissenchaft- 
licher Forschung, Pp. 344, illustrated. Halle, a, S., 1S90. 

Martin, John H. — A manual of microscopic mounting with notes on the collection and exam- 
ination of objects. 2d ed. Illustrated. London, 1878. 

Mason, John J. — Mimite structure of the central nervous system of certain reptiles and 
batrachians of America. Illustrated b}' permanent photo-micrographs. Newport, 1S79-S2. 

Matthews, C. G., and Lott, F. E. — The microscope in the brewery and the malt house. 
Illustrated. London, 1S89. 

Mayall, John, Jr. — Cantor lectures on the microscope, delivered before the society for the 
encouragement of arts, manufacturers and commerce. Nov. -Dec, 1885. History of the micro- 
scope, and figures of many of the forms used at various times. 

Meyer, A. Die Grundlagen und die Methoden fur die mikroskopische Untersuchung von 
Pflanzenpulvern. Pp. 25S, illustrated. Jena, 1901. 

Moeller, J. — Eeitfaden zu mikroskopisch-pharmakgnostischen Ubungen. Pp. 336, illus- 
trated. Wien, 1901. 

Moitessier, A. — Ea photographie appliquee aux recherches micrographiques. Paris, 1866. 

Morel et Soulie. — Manuel de technique microscopique. Paris. 1899. 

Nageli und Schwendener. — Das Mikroskop, Theorie und Anwendung desselben. 2d ed. Pp. 
647. illustrated. Eeipzig, 1877. 

Neuhauss, R. — Eehrbuch der Mikro-photographie. Pp. 266. Illustrated. 2d ed. revised. 
Braunschweig, 189S. 

Nichols, E. E- — The outlines of physics. Illustrated, N. Y., 1897. 

Nichols, E. E. and Franklin W. S. — The Elements of Physics ; Eight and Sound. Pp. 201, 
illustrated. New York and London, 1897. 

Pappenheim, A. — Grundriss der Farbenchemie zum Gebrauch bei mikroscopischen Arbei- 
ten. Pp.476. Berlin, 1901. 

Petri, R. J. — Das Mikroskop von seinen Anfangen bis zur jetzigen Vervollkommung fur alle 
Freunde dieses Instruments. Pp. 24S. Illustrated. Berlin, 1896. 

Phin, J. — Practical hints on the selection and use of the microscope for beginners. 6th ed. 
Illustrated. New York, 1891. 

Posselt, E- A. — The structure of fibres, yarns and fabrics. Illustrated. Philadelphia and 
London, 1891. 

Pre3 _ er, W. — Die Blutkrystalle. Jena, 1S71. Full bibliography to that date. 

Pringle, A. — Practical photo-micrography. Pp. 193, illustrated. New York, 1890. 

Procter, R. A. — The spectroscope and its work. London, 1882. 

Ouekett, J. — A practical treatise on the use of the microscope, including the different methods 
of preparing and examining animal, vegetable and mineral structures. Pp. 515, 12 plates. 2d ed. 
London, 1852. 

Rafter, G. W — The Microscopical Examination of Potable Water. Pp. 160, New York, 1892. 

Ranvier, L- — Traite technique d'histologie. Pp. 1109, illustrated. Paris, 1S75-1888. Structure 
and methods. Also German translation, 1888. 

Reeves, J. E. — A hand-book of medical microscopy, including chapters on bacteriology, neo- 
plasms and urinary examination. Illustrated. Philadelphia, 1894. 



286 BIBLIOGRAPHY 

Reference Hand-Book of the medical sciences. Albert H. Buck, editor. 8 quarto vols. Illus- 
trated with many plates, and figures in the text. New York, 1885-1889. Supplement, 1893. New 
edition, 1901 + . 

Richardson, J. G. — A hand-book of medical microscopy. Pp. 333, illustrated. Philadelphia, 
1871. Methods and descriptions. 

Robin, Ch. — Traite du microscope et des injections. 2d ed. Pp. 1101, illustrated. Paris, 1877. 
Methods and structure. 

Roscoe, Sir Henry. — lectures on spectrum analysis. 4th ed. London, 1885. 

Rosenbusch, K. H. F. translated by Iddings, P. — Microscopical physiography of the rock 
making minerals. Illustrated. New York, 1S89. 

Ross, Andrew. — The microscope. Being the article contributed to the "Penny Cyclopaedia." 
Republished in New York in 1877. Illustrated. 

Rusby, H. H. and Jeliffe, S. E. — Morphology and Histology of Plants designed especially as a 
guide to plant analysis and classification, and an introduction to pharmacognosy and vegetable 
physiol. Pp. 378, illustrated. New York, 1899. 

Rutherford, W. — Outlines of practical histology. 2d. ed. Illustrated. Pp. 194. London and 
Philadelphia, 1876. Methods and structure. 

Sayre, L. E. — Organic Materia Medica and Pharmacognosy. An introduction to the vegetable 
kingdom and the vegetable and animal drugs. Chapters on insects injurious to drugs and on 
pharmacal botany. Pp. 220, Detroit, 1880. 

Schafer, E. A. — A course of practical histology, being an introduction to the use of the micro- 
scope. Pp. 304, 40 Fig. Philadelphia, 1877. Methods. 

Schellen, H. — Spectrum analysis, translated by Jane and Caroline Lassell. Edited with notes 
by W. Huggins. 13 plates, including Angstrom's and Kirchhoff's maps. London 1885. 

Schimper, A. F. W. — Anleituiig zur mikroskopischen Untersuchungen der vegetabilischen 
Genussmittel. Pp. 158, illustrated. Jena, 1900. 

Schneider, A. — Microscopy and micro-technique. Pp. 1S9, illustrated. Chicago, 1899. 

Sedgwick and Wilson. — General biology. 2d ed. Illustrated. New York, 1895. 

Seiler, C. — Compendium of microscopical technology. A guide to physicians and students in 
the preparation of histological and pathological specimens. Pp. 130, illustrated. New York, 18S1. 

Silliman, Benj., Jr, — Principles of ph\-sics, or natural philosophy. 2d ed., rewritten. Pp. 710, 
722 illustrations. New York and Chicago, i860. 

Spitta, E. J. — Photomicrography.* 4 . Pp. 163. Illustrated by half-tone reproductions from 
original negatives. Text illustrations. Loudon, 1S99. 

Starr, Allen M., with the coSperation of Oliver S. Strong and Edward Learning. — An atlas of 
nerve-cells. Columbia University Press, New York, 1896. The atlas consists of text, diagrams, 
and some of the best photo-micrographs that have ever been published. 

Sternberg, G. M. — Photo-micrographs, and how to make them. Pp. 204, 20 plates. Boston, 
18S3. 

Stoehr, S. P.— Text-book of histology including the microscopic technique, 3d American from 
8th German edition. Translated by Dr. Emma Bilstein and edited by Dr. A. Schaper. Pp. 432, 
illustrated. Philadelphia, 1901. 

Stokes, A. — Aquatic microscopy for beginners, or common objects from the ponds and ditches. 
Illustated. Portland, Conn., 1896. 

.Suffolk, W. T. — On microscopical manipulation. 2d ed. Pp. 227, Illustrated. London, 1870. 

Sz3'monowicz, L- — Lehrbuch der Histologie-einschliesslich der mikroskopischen Technik. 
Illustrated. Wtirzburg, 1900. 

Thomas, Mason B., and Wm. R. Dudley. — A laboratory manual of plant histology. Illus- 
trated. Crawfordsville, Ind., 1S94. 

Trelease, Wm. — Poulsen's botanical micro-chemistry ; an introduction to the study of veget- 
able histology. Pp. 11S. Boston, 1SS4. Mehods. 

Trutat, M. — La photographie appliquee a histoire naturelle. Pp. 225, illustrated. Paris, 
1884. 



BIBLIOGRAPHY 287 

Valentin, G. — Die Untersuchung der Pflanzen und der Thiergewebe in polarisirtem Licht. 
Leipzig, 1S61. 

Van Heurck, H — The microscope. Illustrated. London and New York, 1898. 

Vierordt. Die quantitative Spectralaualyse in ihrer Anwendung auf Physiologie, 1876. 

Vogl, A. E. — Die wichtigsten vegetablischen Nahrungs mid Genussmittel. Pp. 575., illus- 
trated. Wien, 1S99. 

Vogel, Conrad. — Practical pocket-book of photography. Pp. 202, Figs. London, 1893. 

Vogel, H. W. — Practische Spectralaualyse irdischer stoffe ; Anleitung zur Benutzung der 
Spectralapparate in der qualitativen und quantitativen chemische Analyse organischer und un- 
organscher Korper. 2d ed. Figs. Berlin 1889. 

Wall, O. A. — Notes on Pharmacognosy. — Pp. 180, illustrated. St Louis, 1897. 

Weinschenk, E. Anleitung zurn Gebrauch des Polarisationsmikroskopes. Pp. 123, illus- 
trated. Freiburg. 1901. 

"Wethered, M. — Medical microscopy. Pp. 406, Figs. London and Philadelphia, 1892. 

"Whipple, G. C. — The Microscopy of Drinking Water. Pp. xii + 300. Illustrated. New York, 
1899. 

Whitman. C. O. — Methods of research in microscopical anatomy and embryology. Pp. 255, 
illustrated. Boston, 1S85. 

Wilder and Gage. — Anatomical technology as applied to the domestic cat. An introduction to 
human, veterinary and comparative anatomy. Pp. 575, 130 Fig. 2d ed. New York and Chicago, 
1SS6. 

Wilson, Edmund, B., with the cooperation of Edward Teaming. — An atlas of fertilization 
and karyokinesis. Columbia University Press, New York, 1895. This atlas marks an era in 
embryological study. It has admirable texts and diagrams, but the distinguishing feature is the 
large number of almost perfect photo-micrographs. 

Wood, J. G. — Common objects for the microscope. Pp. 132. London, no date. Upwards of 
400 figures of pretty objects for the microscope, also brief descriptions and direction for prepa- 
rtions. 

Wormly, T. G. — The micro-chemistry of poisons. 2d ed. Pp. 742, illus. Philadelphia, 1885. 

Wright, Lewis. — Optical Projection, a treatise on the use of the lantern in exhibition and 
scientific demonstration. Pp. 425, illustrated. London, 1891. (Beginners will find this book 
very helpful). 

Wythe, J. H. — The microscopist ; a manual of microscopy and a compendium of microscopi- 
cal science. 4th ed. Pp. 434, 252 Fig. Philadelphia, 1880 

Zimmermann, Dr. A. — Das Mikroskop, ein Leitfaden der wissenschaftlichen Mikroskopie. 
Illustrated. Leipzig und Wein, 1S95. 

See also Watt's chemical dictionary, and the various general and technical encyclopedias. 

PERIODICALS* 

The American journal of anatomy (including histology, embryology and C3'tology). Balti- 
more, 1901-K 

The American journal of medical research, Boston, 1901-!-. 

The American Journal of physiology. Boston, 1S96+. 

The American journal of microscopy and popular science. Illustrated. New York, 1876-18S1. 

The American monthly microscopical journal. Illustrated. Washington, D. C, 1880 + . 

American naturalist. Illustrated. Salem and Philadelphia, 1867+ . 

American quarterly microscopical journal, containing the transactions of the New York 
microscopical society. Illustrated. New York, 1878+. 

American microscopical society, Proceedings. 1878-1894 ; Transactions, 1S95 + . 



*Note — When a periodical is no longer published, the dates of the first and last volumes are 
given ; but if still being published, the date of the first volume is followed by a plus sign. 

See Vol. XVI of the index Catalog of the Library of the Surgeon General's office for a full 
list of periodicals. 



288 BIBLIOGRAPHY 

Anatomischer Anzeiger. Centrablatt fur die gesammte wissenchaftliche Anatomie. Amt- 
liches Organ der anatoniischen Gesellschaft. Herausgegeben von Dr. Karl Bardeleben. Jena, 
1886+ . Besides articles relating to the microscope or histology, a full record of current anatomi- 
cal literature is given. 

Annales de la societe beige de microscopic Bruxelles, 1874 f . 

Archives d' Anatomie microscopique. Illustrated. Paris, 1S97. (Balbiani et Ranvier). 

Archiv fur miroscopische Anatomie. Illustrated. Bonn, 1865 + . 

Centrablatt fur Physiologic Unter Mitwirkung der physiologischen Gesellschaft zu Berlin 
Herausgegeben von S. Exner und J. Gad. Leipzig and Wien. 1SS7 + . Brief extracts of papers 
having a plwsiological bearing. Full bibliography of current literature. 

English mechanic. London, 1S66 — . Contains many of the papers of Mr. Nelson on light- 
ing, photo-micrography, etc. 

Index medicus. New York, 1S79+. Bibliography, including histology and microscopy. 

International journal of micioscopy and popular science. London, 1890+. 

Journal of anatomy and physiology. Illustrated. London and Cambridge, 1867-7-. 

Journal of Applied Microscopy and laboratory methods. Illustrated. Rochester, N. Y., 
1S984-. 

Journal de micrographie. Illustrated. Paris, 1877-1892. 

Journal of microscopy and natural science. London, 1S85 + . 

Journal of the New York microscopical society. Illustrated. New York, 1885-f. 

Journal of physiology. Illustrated. London and Cambridge, 1878-f-. 

Journal of the American chemical society. New York, 1S79 + . 

Journal of the chemical society. London, 1S4S -I- . 

Journal of the royal microscopical society. Illustrated. London, 1878+. Bibliography of 
works and papers relating to the microscope, microscopical methods and histology. It also in- 
cludes a summary of many of the papers. 

Journal of the Ouekett microscopical club. London, 1868 + . 

The Lens, a quarterly journal of microscopy, and the allied natural sciences, with the tran- 
sactions of the state microscopical society of Illinois. Chicago, 1872-1873. 

The Metallographist, a quarterly publication devoted to the study of metals with special 
reference to their physics, microstructure, their industrial treatment and application. Illustrated 
especially by photo-micrographs of metals and alloys. Boston, 1898+. 

The Microscope. Illustrated. Washington, D. C, 18S1-1897. 

Microscopical bulletin and science news. Illustrated. Philadelphia, 1SS3 + . The editor, Ed- 
ward Penuock introduced the term "par-focal " for oculars (see vol. iii, p 31). 

Monthly microscopical journal. Illustrated. London, 1869-1877. 

Nature. Illustrated. London, 1869+. 

The Observer. Portland, Conn., 1S90-1897. 

Philosophical Transactions of the Royal Society of London. Illustrated. London, 1665 + . 

Proceedings of the American microscopical society, 1878+ . 

Proceedings of the Royal Society. London, 1854+. 

Quarterly journal of microscopical science. Illustrated. London, 1S53 + . 

Science Record. Boston, 1883-4. 

Zeitschrift f . Angewandte. Mikroskopie. 1S98+. 

Zeitschrift f ilr Instrumentenkunde. Berlin, 1881-K 

Zeitschrift fiir physiologische Chemic Strassburg, 1S77 + . 

Zeitschrift fiir wissenschaftliche Mikroskpie und fiir mikroskopische Technik. Illustrated. 
Braunschweig. 1SS4+. Methods, bibliography and original papers. 

Besides the above-named periodicals, articles on the microscope or the application of the 
microscope appear occasionally in nearly all of the scientific journals. One is likely to get 
references to these articles through the Jour. Roy. Micr. Soc. or the Zeit. wiss. Mikroskopie. 
Excellent articles on Photo-micrography occur in the special Journals and Annuals of Photog- 
raphy. 



INDEX 



Abbe apertometer, 270-272. 

Abbe camera lucida, 123-132. 

Abbe condenser or illuminator, 45-50. 

Abbe's test-plate, method of using, 268- 
270. 

Aberration, chromatic, 4, 5 ; by cover- 
glass, 55 ; spherical, 4, 5, 268. 

Absorption spectra, 136-139, 145-150. 

Acetelyne light, 37, 51, 229. 

Achromatic condenser, 42-43, 256 ; objec- 
tives, ii, 64 ; oculars, 22. 

Achromatism, 12. 

Actinic focus, 226 ; image, 221. 

Adjustable objectives, 11, 12, 54-57; ex- 
periments, 54 ; and micrometry, 118 ; 
and photo-micrography, 234. 

Adjusting collar, graduation of, 55. 

Adjustment, of analyzer, 151 ; coarse or 
rapid, and fine, 63, Frontispiece ; of 
objective, n, 12, 54; of objective for 
cover-glass, specific directions, 55 ; 
testing, 63. 

Aerial image, 30, 31, 230. 

Air bubbles, 94, 95. 

Albumen fixative, Mayer's, 198. 

Alcohol, absolute, 198 ; ethylic, 198 ; 
methyl, 198, 203. 

Alcoholic dye, staining sections with, 
189. 

Alum solution, 198. 

Amici prism, 134, 140. 

Amplifier, 109. 

Amplification of microscope, 103. 

Analyzer, 141, 151. 

Anastigmatic objective, 208, 212. 

Angle, of aperture, 15, 16 ; of carbons for 
arc lamps, 251-252 ; critical, 54. 

Angstrom and Stokes' law of absorption 
spectra, 138. 

Angular aperture, 15, 16. 

Anisotropic, 152. 

Apertometer, Abbe's, 270. 

Aperture of objective, 15, 21, 270 ; angu- 
lar, 15, 16 ; formula for,' 16-18 ; of 
illuminating cone, 45 ; numerical, 17, 
19, 270 ; numerical of condenser, 44 ; 
significance of, 20. 

Aphengescope, 267. 

Aplanatic cone, 46; objectives, 11; ocu- 
lar, 22 ; triplet, 7. 



Apochromatic condenser, 42 ; objectives, 
12, 64, 214. 

Apparatus and material, 1, 34, 90, 103, 
122, 134, 205, 243 ; for micro-projec- 
tion, 256, 257 ; for photography, 233 ; 
for photo-micrography, 205. 

Appearances, interpretation, 90-102. 

Arc lamp, 37, 229, 250-255 ; continuous 
current, 249. 

Arrangement of condenser, 46 ; of lamp, 
bull's eye and microscope, 51 ; mi- 
nute objects, 204 ; serial sections, 
192 ; tissue for sections, 191. 

Artifacts, 91. 

Artificial illumination, 37, 48, 50 ; for 
photo-micrography, 229. 

Avoidance of diffusion currents, 180 ; of 
distortion, 124. 

Axial light, 36 ; experiments, 40 ; with 
Abbe illuminator, 48. 

Axial, point, 15 ; ray, 36. 

Axis, optic, 2, 3, 10 ; of illuminator, 49 ; 
secondary, 3, 6, 49. 



Back-ground for photographing, 209. 

Back combination or system of objective, 
9-12. 

Bacterial cultures, photographing, 241- 
242. 

Balsam, 199 ; bottle, 183 ; mounting in, 
183, 190 ; preparation of, 199 ; re- 
moval from lenses, 61 ; natural, 190, 
199 ; neutral, 199 ; removal from 
slides, 162 ; xylene, 199. 

Bands, absorption, 137. 

Base of microscope, Frontispiece. 

Bath, water, 255. 

Bed, camera, 207. 

Benzin, removing from sections, 189. 

Biaxial crystals, 154. 

Bibliography, 26, 33, 99, 101, 121, 133, 

150, i55> 157, 158.159. x 9 6 , 204, 220, 
240, 242, 251, 258, 267, 273, 274, 282. 

Blocks for collodion, 178 ; for shell vials, 
174. 

Blood, absorption spectrum of, 146 ; cir- 
culation of with micro-projection, 
257, 264. 

Board, circulation, 280. 

Body of microscope, Frontispiece. 



290 



INDEX 



Bottle for balsam, glycerin or shellac, 

172 ; reagent, 179. 
Bowl, waste, 181. 
Box, glass, 181 ; slide, 198. 
Brownian movement,. 99. 
Bubble, air, 94, 95. 
Bull's-eye, 51, 217, 227 ; engraving glass 

for, 228. 
Burning point, 7, 30. 
Buxton's photo-micrographic outfit, 237- 

238. 



Cabinet for microscopical preparations. 
196,197. 

Calipers, micrometer, 164 ; pocket, 164. 

Camera, bed, 207 ; for embryos, 211 ; for 
large, transparent sections, 215 ; 
photo-micrographic, 222, 225, 235 ; 
testing, 223; vertical, 205-210. 

Camera lucida, Abbe, 122-132; Wollas- 
ton's, 107, 125. 

Canada balsam, 199 ; mounting in, 183, 
190 ; preparation of, 199 ; removal 
from lenses, 61 ; removal from slides, 
162. 

Carbol-xylene, 200. 

Carbon-monoxide hemaglobin, spectrum 
of, 147. 

Carbons, for projection apparatus, 250- 
2 55 ; adjusting, 251 ; angle, 251 ; 
kinds, 253 ; positive and negative, 
252 ; size, 254 ; wear, 253. 

Card, catalog, 196 ; centering, 169 ; inven- 
tory, 281. 

Care of, eyes, 61 ; micro-projection appa- 
ratus, 265 ; microscope, mechanical 
parts, 59 ; optical parts, 60 ; nega- 
tives, 211 ; water immersion objec- 
tives, 58. 

Carmine to show currents and pedesis, 
99 ; spectrum of, 148. 

Carrier, objective, 259. 

Castor-xylene clarifier, 200. 

Cataloging, formula, 195 ; preparations, 
194-196. 

Cedar-wood oil, bottle for, 199 ; for clear- 
ing, 1S4-185, 200; for oil immersion 
objectives, 200. 

Cells, deep, thin, 168 ; isolated, prepara- 
tion of, 175 ; mounting, 168 ; stain- 
ing, 174. 

Cement, shellac, 203. 

Cementing collodion, 201. 

Center, optical, 2, 3. 

Centering, and arrangement of illumina- 
tor, 43, 47 ; card, 169 ; image of source 
of illumination, 44 ; the radiant, 255. 

Centimeter rule, 104, 133. 



Central light, 36, 95. 

Chamber, moist, 171. 

Cheese-cloth for cleaning slides, 161. 

Chemical focus, 12 ; rays, 12. 

Chemistry, Micro-155. 

Chromatic, aberration, 4 ; correction, 11, 
12 ; correction, test for, 270. 

Circulation of blood with micro-projec- 
tion, 257, 264; board, 226. 

Clarifier, 200 ; castor-xylene, 200. 

Class demonstrations in histology and 
embryology, 243. 

Cleaning back lens of objective, 61 ; ho- 
mogeneous objectives, 59 ; micro-pro- 
jection apparatus, 265 ; mixtures for 
glass, 165; sildes and .cover-glasses, 
161-163 ; water immersion objectives, 
58. 

Clearer, 173, 183, 190, 200. 

Clearing mixture, preparation of, 200 ; 
tissues with cedar-wood oil, 184. 

Clinical microscope, 243-245. 

Cloudiness of objective and ocular, how 
to determine, 92 ; removal 60. 

Coarse adjustment of microscope, Front- 
ispiece ; testing, 63. 

Cob-web micrometer, 117. 

Collecting can, 280 

Collodion, 200 ; for coating glass rod, 97 ; 
cementing, 201 ; clarifying, 178 ; cot- 
ton, 200 ; for fastening sections to 
slide, 188 ; hardening, 178 ; method, 
176 ; thick, thin, 177, 201. 

Color, correct photography, 217 ; correc- 
tion, 12 ; images, 54, 58 ; law of, 138 ; 
production of, 154 ; screens, 218, 220. 

Colored minerals, absorption spectra of, 
149 ; specimens, photography of, 218. 

Comparison prism, 141, 142 ; spectrum, 
142. 

Compensating ocular, 24, 25. 

Complementary spectra, 139. 

Compound microscope, see under micro- 
scope. 

Concave lenses, 3 ; mirror, use of, 37. 

Condenser, 41-50 ; Abbe, 46, 48-49 ; ach- 
romatic, 42, 43, 227, 256 ; apochro- 
matic, 42, 227 ; bull's-eye, 51, 227, 
22S ; centering, 43, 47 ; illuminating 
cone with, 45 ; lamp, for projection, 
250 ; mirror for, 48 ; non-achromatic, 
46 ; numerical aperture of, 44-46 ; 
optic axis of, 43, 47 ; for photo-micro- 
graphy, 42, 227, 233 ; standard size 
for, 26, 47 ; substage, 42; See also 
illuminator. 

Condensing lens, 36. 

Cone, aplanatic, 46 ; illuminating, 45. 

Conjugate foci, 4. 

Construction of images, geometrical, 6. 



INDEX 



291 



Continuous, current arc lamp, 249 ; spec- 
trum, 137. 

Contoured, doubly, 97. 

Converging lens, 3-5 ; lens system, 9. 

Convex lenses, 3-5. 

Cooler, specimen, 257. 

Correction, chromatic, or color, 5, 12, 268- 
270 ; cover-glass, 55-56 ; cover, tube- 
length for, 56-57 ; over and under, 12. 

Cotton, collodion, gun, or soluble, 200. 

Counterstaining, 189-190. 

Cover-glass, or covering glass, 162-163 ; 
aberration by, 54 ; adjustment, spe- 
cific directions, 55 ; adjustment for, 
in photo-micrography, 234; adjust- 
ment and tube-length, 12, 13, 55 ; 
anchoring, 170 ; cleaning, 162-163 ; 
correction, 55, 56 ; effect on rays from 
object, 17, 55 ; gauges, 164-165 ; meas- 
urer, 164-165; measuring thickness of, 
163 ; non-adjustable objectives, table 
of thickness, 14; No. 1, variation of 
thickness, 164; putting on, 94, 167; 
sealing, 169, 170; size of, 164, 162; 
thickness of, 13, 14, 163-165, 193 ; 
tube-length with, 13, 56, 57 ; wiping, 
163 ; with, serial sections, 193. 

Crater of carbons, 252. 

Critical angle, 54. 

Crystals, biaxial, depolarizing, 154 ; from 
frog for pedesis, 100. 

Crystallization under microscope, 50, 157. 

Crystallography, 155 ; list of substances 
for, 156-157. 

Currents, diffusion, avoidance of, 180 ; 
in liquids, 98. 

Cutting sections, 178, 186, 191-192. 

D 

Dark-ground illumination, 37, 49-50 ; 
with Abbe illuminator, 50 ; with 
mirror, 50. 

Daylight, lighting with, 35. 

Deck-plugs for collodion blocks, 178. 

Dehydration, 177, 190. 

Demonstration, microscope, 243-245; 
with micro-projection apparatus, 265. 

Depolarizing crystals, 154. 

Designation, of oculars, 25 ; of wave 
length; 143. 

Determination, of, field of microscope, 
28 ; equivalent focus, 272-273 ; mag- 
nification, 103-109, 273 ; of working 
distance, 39. 

Diamond, writing, 196. 

Diaphragms and their employment, 36-50. 

Diffraction, grating, 137 ; illusory ap- 
pearances due to, 101. 

Diffusion currents, avoidance of, 180. 



Direct, light, 35 ; vision spectroscope, 
134- 

Dispersing prism, 137. 

Dissecting microscope, 8, 33, 175, 228. 

Dissociator, formaldehyde, 201 ; nitric 
acid, 203. 

Distance, principal focal, 3. 30 ; standard 
at which the virtual image is meas- 
ured, 109 ; working d. of simple mi- 
croscope or objective, 39 ; working 
d. of compound microscope, 11, 34, 
39-4o. 

Distinctness of outline, 96. 

Distortion in drawing, avoidance of, 124. 

Diverging lens, 3. 

Double spectrum, 142 ; vision, 103, 105. 

Doubly contoured, 97 ; refracting, 152. 

Draw-tube, Frontispiece ; pushing in, 38. 

Drawing, with Abbe camera lucida, 129- 
131 ; board for Abbe camera lucida, 
129, 130 ; distortion, avoidance of, 
124 ; embryograph for, 132 ; with 
microscope, 122 ; photographic cam- 
era for, 132 ; for photo-engraving, 
273 ; scale and enlargement of, 131 ; 
with simple microscope, 132. 

Dry objectives, 10, 16-18 ; light utilized, 
17 ; dry mounting, 167 ; numerical 
aperture, 16 ; dry plates, discovery by 
Maddox, 221. 

Dust, of living rooms, examination of, 
101 ; on objectives and oculars, how 
to determine, 92 ; removal, 60. 

Dye, general, staining with, 189 ; aque- 
ous, 180, 189 ; alcoholic, 180, 189. 

E 

Eccentric diaphragm, 44, 49-50. 

Egg pippett, 280. 

Electric light, 37, 229, 235, 250. 

Embryograph, 132 

Embryos, camera for, 211 photograph- 
ing, 211-214; records of, 213. 

Engraving glass for bull's-eye condenser, 
228 

Enlargements, 241. 

Eosin, 2or. 

Epidiascope, 266-267. 

Episcope, 267. 

Equivalent focal length or focus of objec- 
tives and oculars, 10, 25, 272. 

Erect image 1. 

Ether, alcohol, 201 ; sulphuric, 201. 

Ethylic alcohol, 19S. 

Examination of dust of the living rooms, 
bread crumbs, corn starch, fibres of 
cotton, linen, silk, human and ani- 
mal hairs, potatoes, rice, scales of 
butterflies and moths, wheat, 101. 



292 



INDEX 



Experiments, Abbe condenser, 48 ; with 
adjustable and immersion objectives, 
54 ; compound microscope, 26 ; ho- 
mogeneous immersion objective, 58 ; 
lighting and focusing, 37 ; in micro- 
chemistry, 157 ; with micro spectro- 
scope, 145 ; with micro-polariscope, 
152 ; in mounting, 166 ; photo-micro- 
graphy, 229 ; simple microscope, 6. 

Exposure, of photographic plates, 232, 
235, 240; with color-screen 220. 

Extraordinary ray of polarized light, 150. 

Eye and microscope, 6, 9, 32. 

Eyes, care of, 61 ; muscae volitantes of, 
100. 

Eye-lens of the ocular, 22. 

Eye-piece, 22 ; micrometer, 1 14. 

Eye-point, 7, 22, 123 ; of ocular, demon- 
stration, 32. 

Eye-shade, adjusting, 62 ; double, 62. 



Farrants' solution in mounting objects, 
171 ; preparation of, 201. 

Fibers, examination of, 101 ; textile, 158. 

Field, 28 ; with camera lucida, 107 ; illu- 
mination of, 45, 51 ; with orthoscopic 
ocular, 23 ; with periscopic ocular, 
24 ; of view with microscope, 28, 29, 
105, 123-125; size of, with different 
objectives and oculars, 28, 29. 

Field-lens, of ocular, 22 ; action of, 32 ; 
dust on, 92. 

Filar, micrometer ocular, 23, 26 ; ocular 
micrometer, 117, 118. 

Filter, hot, 1S5. 

Filtering balsam, etc., 185, 199. 

Fine adjustment, Frontispiece ; testing, 

6 3- 
Fir, balsam of, 199. 

Fixative, albumen, Mayer's, 198. 

Fixing, reagents for, 19S ; tissue, 176. 

Focal distance, or point, principal, 30 ; 
length equivalent, 10. 

Focus, 6; actinic, 226; chemical, 12 ; 
conjugate, 4 ; equivalent, of object- 
ives and oculars, 10, 25, 272-273 ; op- 
tical, 12 ; principal^, 5 ; virtual, 3 ; 
visual, 226. 

Focusing, 6, 34 ; adjustments, testing, 63; 
with compound microscope, 34 ; em- 
bryos, 213 ; experiments, 38 ; glass, 
209 ; with high objectives, 38 ; with 
low objectives, 38 ; objective for mi- 
cro-spectroscope, 144 ; for photo-mi- 
crography, 206, 213, 230; scale for, 
206 ; screen for photo-micrography, 
209 ; with simple microscope, 6, 34 ; 
slit of micro-spectroscope, 145. 



Food, detection of adulteration in, 158. 

Form of objects, determination of, 93. 

Formal, 201. 

Formaldehyde, dissociator, 201 ; for isola- 
tion, 173. 

Formula, for aperture, 16, 17 ; for catalog- 
ing* J 95 I Ior refraction, 52-54. 

Fraunhofer lines, 137. 

Free, hand sections, 274 ; working dist- 
ance, 40. 

Front combination or lens of objective, 
9-1 1. 

Frontal sections, 192. 

Function of objective, 29-31 ; of ocular, 



Gauge, cover-glass, 164-165. 

Gelatin, liquid, preparation of, 203. 

Geometrical construction of images, 6. 

Glass, cleaning mixture for, 165 ; focus- 
ing, 209 ; ground, 29, 209 ; rod appear- 
ance under microscope, 96, 97 ; slides 
or slips, 161, 162. 

Glasses, opera, 262. 

Glue, liquid, preparation of, 203. 

Glycerin, bottle for, 183 ; mounting ob- 
jects in, order of procedure, 170, 201 ; 
removal, 61. 

Glycerin jelly, mounting objects in, or- 
der of procedure, 170-171 ; prepara- 
tion of, 201. 

Grating, diffraction, 137. 

Ground glass, focusing screen, 209 ; pre- 
paration of, 29. 

Gun cotton, 200. 

H 

Half-tones from photo-micrographs, 232, 

274. 
Hardening collodion, 178 ; tissue, 176, 

183. 
Hematein, 203. 
Hematoxylin, 202 ; stained preparations, 

photographing, 218. 
Hemoglobin spectrum, 147. 
High school microscope, 64, 71-89. 
Histology, physiological, 196. 
History of photo- micrography, 220. 
Holder, lens, 7, 217, 228 ; needle, 167 ; 

slide, 188. 
Homogenous immersion objective, 16- 

19 ; cleaning, 59 ; experiments, 58 ; 

numerical aperture, 16-21. 
Homogenous liquid, 11 ; tester for, 58, 

271 ; vessel for, 199. 
Horizontal camera, 230, 236-237. 
Huygenian ocular, 22, 24, 32. 






INDEX 



293 



Illuminating, cone (for condenser), aper- 
ture of, 45 ; objective, 13, 238 ; 
power, 21. 

Illumination, for Abbe camera lucida, 
129 ; artificial, 37, 48, 50 ; artificial 
for photo-micrography, 229 ; center- 
ing image of source of, 44 ; with air 
and oil, 94, 95 ; dark ground, 49, 
50 ; daylight, 35 ; of entire field, 51 ; 
lamp for, 50 ; methods of, 35, 49 ; for 
micro-polariscope, 151 ; for micro- 
spectroscope, 144 ; oblique with air 
and oil, 94, 95 ; of opaque objects, 
144, 238 ; for photography, 208, 
216, 229, 241 ; for photo-micrography, 
230, 233, 238 ; for projection, 250 ; for 
Wollaston's camera lucida, 124. 

Illuminator, 41-50; vertical, 13, 238. 
See also condenser. 

Image, actinic, 221 ; aerial, 30, 230 ; center- 
ing i. of source of illumination, 44 ; 
color, 52, 58 ; inverted, real of ob- 
jective, 30 ; of flame, 45 ; geomet- 
rical construction of, 6 ; and object, 
size and position, 5, 9, 108 ; real, 5, 9, 
30-32, 103 ; refraction, 52, 58 ; retinal, 
6, 9, 32 ; swaying of, 48 ; virtual i. 
and standard distance at which meas- 
ured, 6, 109. 

Image-power of objectives, iS. 

Imbedding, 177, 185. 

Immersion, fluid or liquid, 58, 271 ; illu- 
minator, 47 ; objective, 11, 58-59. 

Incandescence or line spectra, 137. 

Incident light, 35. 

Index, of refraction, 53 ; of medium in 
front of objective, 16-19. 

Indicator ocular, 247. 

Infiltration, collodion, 177 ; paraffin, 184; 
paraffin dish for, 279. 

Ink for labels and catalogs, 195 ; for 
drawing, 274. 

Interpretation of appearances under the 
microscope, 90-102. 

Inventory card, 281. 

Iris diaphragm, 82, 157. 

Irrigating with reagents, 170. 

Isochromatic plates, 217. 

Isolation, 173 ; with formaldehyde, 173 ; 
nitric acid, 175. 

Isotropic, 152. 

J-K 

Japanese filter or tissue paper, 60. 
Jelly, glycerin, 170-171, 201. 
Jena glass, 71. 

Jurisprudence, micrometry in, 121. 
Knife support, 277. 



Labels and catalogs, 194-196, 203. 

Labeling microscopical preparations, 194; 
photographic negative, 211 ; serial 
sections. 193. 

Laboratory compound microscope, 64, 
71-89. 

Lamp, acetylene, 37, 5T, 229 ; condenser, 
250 ; electric arc, 37, 229, 250-255 ; 
petroleum, 37, 50-51, 220, 229 ; spirit, 
183. 

Lantern, magic, 249 ; slides, 241. 

Law of color, 138. 

Lens, concave, 3 ; converging, 3 ; convex, 
4 ; eye, 22 ; field, 32 ; holder, 7, 175, 
217, 22S ; paper, 60 ; system, 9 ; 
thick, 3. 

Lenses of micro-projection apparatus, 
cleaning, 265. 

Letters in stairs, 93 ; for photo-engraving, 
274. 

Lettering oculars, 26. 

Light, with Abbe illuminator, 48 ; ace- 
tylene, 37, 51, 229 ; artificial, 37, 50, 
229 ; axial, 36, 40, 48 ; direct, 35 ; 
central, 36, 40 ; electric, 37, 229, 250 ; 
incident, 35 ; with mirror, 37 ; ob- 
lique, 36, 41, 48 ; petroleum, 37, 50, 
220, 229 ; for photo-micrography, 229; 
polarized, 150; reflected, 35; sun, 
229; transmitted, 36; utilized with 
different objectives, 17; for vertical 
illuminator, 239 ; wave length of, 
142 ; Welsbach, 37, 229. 

Lighting, 35 ; for Abbe camera lucida, 
129 ; artificial, 50; experiments, 37 ; 
with horizontal camera, 230 ; for mi- 
cro-polariscope, 151 ; for micro-spec- 
troscope, 144 ; with a mirror, 37, 40 ; 
with daylight, 35, 229 ; for photog- 
raphy, 208, 216 ; for photo-micro- 
graphy, 229, 230 ; for vertical illum- 
inator, 229. 

Line spectrum, 137. 

Liquid, currents in, 98 ; homogenous, 11, 
5S. 271. 

M 

Magic lantern, 249. 

Magnification of compensating oculars, 
24 ; effect of adjusting objective, 118 ; 
determination of, 103-109 ; expressed 
in diameters, 103 ; initial or indepen- 
dent, 273 ; of microscope, 103 ; in 
micro-projection, table, 262 ; of mi- 
croscope with Abbe camera lucida, 
131 ; of microscope, compound, 105 ; 
of microscope, simple, 104 ; of photo- 



294 



INDEX 



micrographs, determination of, 233 ; | 
real images, 103 ; table of, with ocu- 
lar micrometer, in : with projection 
microscope, 262 ; varying with com- 
pound microscope, 109; and velocity, 
98. 

Magnifier, tripod, 7, 104, 209. 

Marker for preparations, 65-66. 

Marking objects, 65-66,248 ; negatives, 
211, 214; objectives, 71. 

Masks for preparations, 264. 

Material and apparatus, 1, 34, 90, 103, 
123, I34,_ 156, 198, 205, 223, 243. 

Measure, unit of, in micrometry, 112 ; of 
wave length, 143. 

Measurer, cover-glass, 164-165. 

Measuring the thickness of cover-glass, 
163. 

Mechanical parts of compound micro- 
scope, 64 ; Frontispiece, 8 ; of micro- 
scope, care of, 59 ; testing, 63. 

Mechanical stage, 65, 67-70, 258, 259. 

Megascope, 267. 

Metallic surfaces, photography of, 235- 
240 ; preparation of, 239. 

Metallography, microscope in, 159. 

Metals, examination of, 159, 235. 

Met-hemaglobin, spectrum of, 136, 147. 

Methods, collodion, 176-183 ; paraffin, 
183-191. 

Metric measures and equivalents, cover 
istp., 133. 

Micro-chemistry, 155-157 ; slides for, 161. 

Micro-metallography, objects for, 238. 

Micrometer, 103 ; calipers, 164 ; cob-web, 
117; filar m. ocular, 117, 11S; filling 
lines of, 106 ; lines, arrangement of 
ocular and stage, 120 ; lines, finding, 
106 ; net, 12S ; object or objective, 
105; ocular or eye-piece, 1 14-120; 
ocular, micrometry with, 116; ocular, 
ratio, 119; ocular, valuation of, in, 
114; ocular, varying valuation of, 
118 ; for photo-micrography, 233 ; 
screw ocular, 117; stage, 105, 106; 
table of magnification, in. 

Micrometry, definition, 112, 114; with 
adjustable objectives, 118; compari- 
son of methods, 1 19-120; with com- 
pound microscope, 112; and juris- 
prudence, 121 ; limit of accuracy in, 
120; with ocular micrometer, 116 ; 
with simple microscope, 112 ; remarks 
on, 11S ; unit of measure in, 112. 

Micro-millimeter, 113. 

Micron, 113 ; for measuring wave length 
of light, 143. 

Micro-photograph, 220. 

Micro-photography, distinguished from 
photo-micrography, 220. 



Micro-planar objective, 212, 260. 

Micro-polariscope, 100, 150-155. 

Micro-polarizer, 150. 

Micro-projection, 249-267 ; apparatus, 
256-257 ; carbons for, 250-254 ; cir- 
culation of blood with, 257, 264 ; con- 
denser, 254, 256 ; current, 254 ; dem- 
onstration with, 265 ; magnification, 
262 ; masks for specimens, 264 ; me- 
chanical stages, 258-259 ; microscope 
for, 249 ; objectives and oculars, 259 ; 
pointer for, 265 ; preparations, 263 ; 
screen and screen distance, 261 ; 
specimen cooler, 257 ; stage, 258 ; 
stains for, 263 ; water-bath, 255. 

Microscope, definition, 1 ; amplification 
of, 103 ; clinical, 243 ; demonstration, 
243 ; dissecting, 8, 33, 175, 176 228; 
care of, 59; eye and, 1, 6, 9; field of, 28, 
29 ; focusing, 34 ; magnification, 103; 
for metallography, 159-160 ; for mi- 
crochemical analysis, 153; for opaque 
objects, 235-237 ; for photo-micro- 
graphy, 225, 231, 237 ; polarizing, 150; 
preparation, with erecting prism 176; 
projection, 249-250; price of, 64, 71 ; 
putting an object under, 27 ; sun or 
solar, 249 ; screen, 59 ; stand for 
large, transparent objects, 215, 216 ; 
stand, for embryos, 212 ; traveling, 
245-246. 

Microscope compound, definition, 8 ; 
drawing with, 122 ; figures, frontis- 
piece, 9, 71-89, 102, 153, 160, 237, 
244-246 ; focusing, 38-39 ; for High 
schools, 64, 71 ; for laboratory, 64, 
71-89; lamp for, 50; magnification 
or magnifying power, 105 ; magnifi- 
cation and size of drawing with 
Abbe camera lucida, 131 ; mechani- 
cal parts, 64 ; micrometry with, 112 ; 
optic axis of, 9, 10 ; optical parts of, 
9, 64 ; varying magnification, 109 ; 
working distance of, 39 ; testing, 63. 

Microscope, simple, definition, 1 ; exper- 
iments with, 6 ; figures, 6-8, 33, 104, 
175, 209, 228, 243 ; focusing with, 34; 
magnification of, 104 ; micrometry 
with, 112; working distance of, 34; 

Microscopic, objective, 9 ;obj ective low, 
attached to camera, 214; objects, 
drawing, 122 ; ocular, 22 ; slides or 
slips, 161 ; tube-length, 13, 14. 

Microscopical preparations, cabinet for, 
197 ; cataloging, 194 ; labeling, 194 ; 
mounting, 166-193. 

Microtome, hand, 274-275 ; Minot's 275- 
276 ; razor support for, 276-277 ; slid- 
ing, 278. 



INDEX 



295 



Micro-spectroscope, 134-150; adjusting, 
139 ; experiments, 145 ; focusing, 
144 ; focusing the slit, 139 ; lighting 
for, 144 ; objectives to use with, 144 ; 
reversal, apparent, of colors in, 134 ; 
slit, mechanism of, 135, 139. 
Micrum, 113. 
Mikron, 113. 

Milk globules, to overcome pedesis 
of, 100 

Minerals, absorption spectra of, 149. 

Minot's microtomes, 275-276. 

Minute objects, arrangement of 204. 

Mirror, 9-1 1 ; for Abbe illuminator, 48; 
of camera lucida, arrangement for 
drawing, 126; concave, use of, 37; 
dark ground illumination, 50 ; light 
with, central and oblique, 40, 41 ; 
lighting with, 37 ; plane, use of, 37. 

Mixture, clearing, 200. 

Models, wax, 274. 

Moist, chamber, 171. 

Molecular movement, 99. 

Monazite sand, spectrum of, 149. 

Mounting cells, preparation of, 168 ; me- 
dia and preparation of, 198 203 ; ob- 
jects fur polariscope, 151 ; perma- 
nent, 167 ; temporary, 166. 

Mounting objects, dry in air, order of 
procedure, 167-168 ; in glycerin, or- 
der of procedure, 170 ; in glycerin 
jelly, order of procedure, 170; in 
media miscible with water, 169 ; mi- 
nute objects, 204 ; opaque objects, 
239 ; permanent, 167 ; in resinous 
media, by drying or desiccation or- 
der of procedure, 172 ; in resinous 
media, by successive displacements, 
order of procedure, 172 ; temporary, 
166. 

Movement, Brownian, or molecular, 99. 

Muscae volitantes, 100. 

Muscular fibers, isolation of, 175. 

N 

Natural balsam, 199. 

Needle-holder, 167. 

Negatives, labeling, 21 r, 214; oculars, 
22 ; rack for drying, 240 ; record of, 
2 r4 ; storing, 2 r r , 2 r4. 

Net micrometer, 128. 

Neutral balsam, 199. 

Nicol prism, T50. 

Nitric acid, dissociator, 203. 

Nomenclature of objectives, 10. 

Non-achromatic condenser, 46 ; object- 
ives, 11. 

Non-adjustable objectives, 12 ; thickness 
of cover glass for, table, 14. 



Normal salt solution, 203. 

Nose-piece, 27, 38, 80 ; marking object- 
ives on, 71. 

Numerical aperture, of condenser, 44 ; of 
objectives, 16, 270 ; table of, 19. 



Object, determination of form, 93 ; hav- 
ing plane or irregular outlines, rela- 
tive position in a microscopical prep- 
ration, 92 ; and image, size of, 10, 
108 ; marking parts of, 65-66 ; mark- 
ing position of, 248 ; micrometer, 105; 
mounting, 166 ; putting under micro- 
scope, 27 ; shading, 59 ; suitable for 
photo-micrography, 227 ; transparent 
with curved outlines, relative posi- 
tion in microscopic preparations, 94. 

Objective, 9-13, 121 ; achromatic, 11 ; 
adjustable, 11, 12, 54; adjustable, 
micrometry with, 118; adjustable, 
photo-micrography with, 234 ; adjust- 
ment for, 54 ; aerial image of, 30 ; 
anastigmat, 208 ; aperture of, 15-22, 
270; aplanatic, 11; apochromatic, 

12, 224 ; back combination of, 10, 11 ; 
carrier, 259 ; cleaning back lens of, 
61 ; collar, graduated for adjustment, 
56 ; cloudiness or dust, how to deter- 
mine, 92; designation of. 10; dry, 
10, 1 7- J 9, 121 ; equivalent focus of, 
10, 25, 29, 272 ; field of, 28-29 ! focus- 
ing for micro-spectroscope, 144 ; front 
combination of, 10, 11 ; function of, 
29-31; glass for, 11-13, 71; high, 
focusing with, 38 ; homogeneous im- 
mersion, 17-19, 1-2 i ; homogeneous 
immersion, cleaning, 59; homogene- 
ous immersion, experiments, 58 ; il- 
luminating, 13, 238 ; image, power 
of, 18 ; immersion, n, 121 ; index of 
refraction of medium in front of, 17, 
19 ; initial or independent magnifica- 
tion of, 273 ; inverted, real image of, 
30 ; for laboratory microscope, 64 ; 
lettering, 10 ; light utilized with, 17 ; 
low, focusing with, 3S ; magnifica- 
tion of, 272 ; marking, by Krauss' 
method, 71 ; for micrometallography, 

13, 159; micro-planar, 212, 260; to 
use with micro-polariscope, 151 ; mi- 
croscopic, 9 ; to use with micro- 
spectroscope, 144 ; for micro-spectro- 
scope, focusing, 144 ; nomenclature 
of, 10; non-achromatic, 11; non- 
adjustable, 12; non-adjustable, thick- 
ness of cover-glass for, table, 14 ; 
with nose piece, 38 ; numbering, 10 ; 
numerical aperture, 16-22, 270 ; oil 



296 



INDEX 



immersion, 11 ; panto-chromatic, 13 ; 
para-chromatic, 13 ; for photography, 
208, 212, 214, 224; for photo-microg- 
raphy, 224; projection, 13, 212, 214, 
2 59 ; putting in position and remov- 
ing, 26 ; semi-apochromatic, 13; table 
of field, 29 ; terminology of, 10 ; un- 
adjustable, 12; variable, 13; visual 
and actinic foci of, in photo-microg- 
raphy, 226 : water immersion, 17-19, 
56 ; working distance of, 39-40. 

Oblique light, 36, 41 ; with Abbe illumi- 
nator, 48; with a mirror, 41, 50. 

Ocular, various forms, 22-25 \ cloudiness, 
how to determine and remove, 60, 92; 
equivalent focus of, 25, 29, 273 ; eye- 
point of, 32 ; field-lens, 32 ; filar or 
screw micrometer, 26, 117; focus, 
equivalent of, 25, 273 ; function of, 
31-33 ; indicator, 67, 247 ; iris dia- 
phragm for, 157 ; lettering and num- 
bering, 26 ; micrometer, micrometry 
with, 114-121 ; parfocal, 24, 38; for 
photo-micrography, 224, 232, 235 ; 
pointer, 247 ; projection, 260 ; spec- 
troscopic. 134 ; standard size for, 26; 
table, effect on field, 29. 

Oil, and air, appearances and distinguish- 
ing optically, 95 ; removal, 61 ; re- 
moval from sections, 179. 

Oil-globules, with central and oblique 
illuminations, 95. 

Oil immersion objectives, n. 

Opaque objects, lighting, 144, 23S ; pho- 
tography of, with microscope, 235- 
240 ; projection of, 267. 

Opera glasses, 262. 

Optic axis, 2, ; of condenser or illumi- 
nator, 47 ; of microscope, 10. 

Optical, bench, 237 ; center, 2 ; focus, 12 ; 
parts of compound microscope, 9, 64; 
parts of microscope, care of, and test- 
ing, 60, 63 ; section, 98. 

Order of procedure in mounting ob- 
jects dry or in air, 167 ; in glycerin, 
170; in glycerin jelly, 170: in resin- 
nous media by desiccation, 172 ; in 
resinous media by successive dis- 
placement, 172. 

Ordinary ray, with polarizer, 150. 

Orthochromatic plates. 217. 

Orthoscopic ocular, field with, 2S. 

Outline distinctness of, 96. 

Oven paraffin, 279. 

Over-correction, 5. 

Oxy-hemoglobin, spectrum of, 138, 147. 



Paper, bibulous, filter, lens, or Japanese 
for cleaning oculars and objectives, 
60, 1 So. 

Paraffin, 185, 203 ; filtering, 185 ; infil- 
trating with, 185 ; dish for infiltrat- 
ing, 279 ; imbedding in, 185, 186 ; 
method, 183 ; oven, 279 ; pail for 
melting, 184 ; removal from lenses, 
61 ; removing from sections, 18S. 

Parfocal oculars, 24, 3S. 

Parts, optical and mechanical of micro- 
scope, 8, 64 ; testing, 63. 

Pedesis, 99 ; compared with currents, 99 ; 
to overcome, 100 ; with polarizing 
microscope, 100 ; proof of reality of, 
100. 

Penetrating power, 21. 

Penetration of objective, 21. 

Permanent mounting, 167 ; preparations 
of isolated cells, 175. 

Permanganate of potash, absorption spec- 
trum of, 136, 146. 

Petri dish, photographing bacterial cul- 
tures in, 241. 

Petroleum light, 37, 229 ; as color screen, 
220. 

Pharmacological products, examination 
of, 158. 

Photo-engraving, drawing for, 273 ; let- 
tering for, 274. 

Photographic, camera, 207 ; negatives, 
labeling, 211, 214; objectives, 208; 
prints, 211. 

Photography, back-ground for, 209 ; of 
bacterial cultures, 241-242 ; color- 
correct, 217 ; of colored objects, 218 ; 
compared with photo-micrography, 
222; of embryos, 211-214; focusing 
and exposure, 206, 213 ; indebtedness 
to photo-micrography, 220; of large 
transparent objects, 214-216; lighting 
for, 20S, 216; metallic objects, 235- 
240 ; objectives for, 208, 212, 214 ; of 
objects in alcohol or water, 206 ; 
opaque objects, 235-240 ; plates for, 
217; stage for, 211 ; with vertical 
camera, 205-209, 225. 

Photo-micrograph, 220 ; determination 
of magnification for, 233 ; at 5-20 di- 
ameters, 212; 20-50 diameters, 230;' 
100-2500 diameters, 233 ; of metallic 
surfaces, 235-240 ; objects suitable 
for, 227 ; of opaque objects, 235-240; 
prints of, 211 ; plates for, 217 ; repro- 
ductions of, 232 ; with and without 
an ocular, 230-234. 

Photo-micrographic, camera, 222, 225, 
236 ; outfit, 236-237 ; stand, 231. 



INDEX 



297 



Photo-micrography, 220-240 ; cover-glass 
correction, 234 ; apparatus for, 223 ; 
compared with ordinary photogra- 
phy, 222 ; condenser for, 42, 226, 227; 
distinguished from micro-photogra- 
phy, 220 ; experiments, 229 ; expos- 
ure for, 213, 220, 232, 235, 240 ; focus- 
ing for, 213, 216 ; focusing screen for, 
209 ; lighting, 229, 230, 233, 239 ; mi- 
crometer for, 233 ; objectives and oc- 
ulars for, 13, 224, 232,235 ; vertical 
camera with, 211, 222, 225 ; actinic 
foci in, 226 ; with and without ocular, 
230,232, 234 ; record table for, 219. 

Physiological histology, 196. 

Picric-alcohol, 203. 

Picro-fuchsin, 190. 

Pillar of microscope, Frontispiece. 

Pin-hole diaphragm, 47. 

Pippett, 179 ; egg, 280. 

Plane mirror, use of, 37. 

Plates, color-correct, 217 ; exposure of, 
213, 220, 232, 235. 240 ; isochromatic, 
or orthochromatic, 217 ; size of, 224. 

Pleochroism, 152. 

Pleurosigma angulatum, 41. 

Point, axial, 15 ; burning, 7. 

Pointer ocular, 247. 

Polariscope, 140, 150. 

Polarized light, extraordinary and ordi- 
nary ray of, 150. 

Polarizer and analyzer, 140, 15 r. 

Polarizing microscope, pedesis with, 100. 

Position of objects or parts of same ob- 
ject, 92 ; marking p., 248. 

Positive oculars, 10, 22. 

Power, of microscope, 103 ; illuminating, 
penetrating, resolving, of objective, 
19-21 ; of ocular, 25. 

Preparation of Canada balsam, Farrant's 
solution, glycerin, glycerin jell}-, etc., 
198-204. 

Preparation, of clearing mixture, liquid 
gelatin and shellac cement, 198-204 ; 
of ground glass, 29 ; of metallic sur- 
faces, 239 ; vials, 174. 

Preparations, cataloging, 194-196 ; cabi- 
net for, 196-197; labeling, 194; for 
microprojection, 263 ; permanent, of 
isolated cells, 175. 

Price of American and foreign micro- 
scope, 71. 

Principal, focus, 3, 5 ; focal distances, 3, 
30 ; optic axis, 2, 5. 

Prism of Abbe camera lucida, 124, 127; 
Amici, 140; comparison, 141; dis- 
persing, 141; erecting, 176; Nicol, 
150 ; and slit of micro-spectroscope, 
mutual arrangement, 139 ; of Wollas- 
ton's camera lucida, 125. 



Prints, photographic, 211. 

Projection, apparatus, 249, 263-297 ; mi- 
croscope, 249-266 ; see micro-projec- 
tion ; objective, 13, 214, 259 ; ocular, 
24, 25, 232, 260 ; opaque objects, 267 ; 
in photo-micrograph}', 232, 234. 

Putting, on cover-glass, 167 ; an object 
under microscope, 27 ; an objective 
and ocular in position, 26, 27. 

Pyroxylin, 200. 

Q-R 

Quadrant for camera lucida, 127, 128. 

Radiant, centering, 255. 

Ratio, ocular micrometer, 119. 

Razor and support, 276-277. 

Reagent, bottle, 179 ; for fixing, 198; irri- 
gation with, 170 ; for mounting, 198. 

Real image, 5, 8, 9, 30; magnification, 103, 
262. 

Record, of negatives, 214; table for photo- 
micrography, 219. 

Reflected light. 35. 

Reflection, total, 54. 

Refraction, 52 ; images, 52, 58 ; index of, 
53 ; of medium in front of objective, 

19- 

Refractive, doubly, 152 ; highly, 97 ; 
singly, 152. 

Relative position of objects, 92. 

Resinous media, mounting objects in, 
order of procedure, by drying or 
desiccation, 172 ; by a series of dis- 
placements, 172. 

Resolution and numerical aperature, 20. 

Resolving power, 20. 

Retinal image, 6, 9. 

Revolving nose-piece, marking objectives 
on, 71. 

Ribbon sections, 186-187 ; tra Y f° r > X S7- 



Sagittal sections, 192. 

Salicylic acid, crystallization, 50. 

Salt solution, normal, 203. 

Scale, of drawing, 131 ; of sizes for pho- 
tographing, 206 ; of wave lengths, 
142. 

Screen, color, 218-219 ; focusing s. for 
photo-micrography, 209 ; of ground 
glass, 29; for micro-projection, 261 ; 
for microscope, 59. 

Screw, society, 64 ; micrometer, 26, 117. 

Sealing cover-glass, 169, 170. 

Searching ocular, 24. 

Secondary axis, 3. 

Section, lifter, 181-182 ; optical, 9S. 

Sections, arrangement of tissue for, 191 ; 
clearing, 190 ; cutting, 178, 186; de- 



298 



INDEX 



h3'dration of, 190 ; extending with 
water, 1S6 ; fastening to slide, 179, 
187 ; frontal, 192 ; mounting, 183, 
190 ; removing benzine, oil and para- 
ffin from, 179, 1S8 ; ribbon, 186 ; 
sagittal, 192 ; serial, 191-193 ; stain- 
ing, 180, 189 ; transferring, 179 ; 
thickness for micro-projection, 263. 

Selenite plate for polariscope, 154. 

Semi-apochromatic objective, 13. 

Serial sections, 191-193 ; arranging and 
labeling, 192, 193 ; stage for, 67, 69 ; 
thickness of cover-glass for, 193. 

Shell vials, 174. 

Shellac cement, preparation of, 203 ; re- 
moval from lenses, 61. 

Significance of aperture, 20. 

Simple microscope, see under microscope. 

Sines, table of, 3d page of cover. 

Slides, 161 ; box for 198; cleaning, 161 ; 
holder for, 188-189 ! lantern, 241 ; for 
micro-chemistry, 161 ; tray for, 1S7. 

Sliding microtome, 278. 

Slips, 161. 

Slit mechanism of micro-spectroscope, 

135, 139- 

Society screw, 64. 

Sodium, lines and spectrum, 136-137. 

Solar spectrum or s. of sunlight, 136-137. 

Soluble cotton, 200. 

Solution, alum, 198 ; Farrants', 201. 

Specimen cooler, 257. 

Spectral, colors, 138 ; ocular, 134, 139. 

Spectroscope, direct vision, 134, 145. 

Spectroscopic, examination of color- 
screens, 220 ; ocular, 134. 

Spectrum, 136-150; absorption, 137; 
amount of material necessary and its 
proper manipulation, 145 ; analysis, 
150 ; Angstrom and Stokes' law of, 
13S ; banded, not given by all colored 
objects, 148 ; of blood, 146 ; of carbon 
monoxide hemaglobin, 147 ; of car- 
mine solution, 148 ; of minerals, 149 ; 
of colorless bodies, 14S ; comparison, 
142 ; complementary, 139 ; continu- 
ous, 137 ; double, 142 ; incandescence; 
137 ; line, 137 ; met-hemaglobin, 147, 
monazitesand, 149 ; oxy-hemoglobin, 
138, 147; permanganate of potash, 136, 
146 ; single-banded of hemaglobin, 
138, 147 ; sodium, 136, 137 ; solar, 
!36, 137 ; two-banded of oxy-hema- 
globin, 147. 

Spherical aberration, 4, 5 ; test for, 268. 

Stage, Frontispiece, 65 ; mechanical, 65, 
67-70, 259 ; micrometer, 105 ; for mi- 
cro-projection, 258 ; for photograph- 
ing, 211. 



Stain, alcoholic, 180, 189 ; aqueous, 180, 
189 ; for micro-projection, 263. 

Staining, cells, 174 ; dish, 190 ; sections, 
180, 189. 

Stand, of microscope, 65 ; photo-micro- 
graphic, 231 ; special for embryos, 
212 ; special for large transparent ob- 
jects, 215, 216. 

Standard, distance (250 mm.) at which 
the virtual image is measured, 109 ; 
screw, 64 ; size for condenser, 47 ; 
size for oculars, 26. 

Stokes and x\ngstrom's law of absorption 
spectra, 138. 

Storing negatives, 211 ; preparations, 196. 

Substage, 86, Frontispiece. 

Substances for crystallography, 156. 

Sulphonal with polarizer, 154. 

Sulphuric ether, 201. 

Support for knife of microtome, 276-277. 

Swaying of image 4S. 

Synthol, 19S. 

System, back, front, intermediate of 
lenses, 10, 11 ; crystal, 156 ; metric, 
cover 1st p., 133. 



Table, for immersion fluid, 272 ; of mag- 
nification and valuation of ocular mi- 
crometer, in ; magnification with 
projection microscope, 262 ; of tube- 
length and thickness of cover-glasses, 
14 ; natural sines, third page of cover; 
of numerical aperture, 19 ; record, 
for photo-micrography, 219 ; size of 
fields, 29 ; testing homogeneous liq- 
uids. 272 ; of valuations of ocular 
micrometer, 111 ; weights and meas- 
ures, 2d page of cover. 

Temporary mounting, 166. 

Terminology of objectives, 10. 

Test of chromatic and spherical aberra- 
tion, 268-270. 

Tester, cover-glass, 164-165 ; for homo- 
geneous liquids, 58, 271. 

Testing a camera, 223 ; a microscope and 
its parts, 63. 

Test-plate, Abbe's, method of using, 268. 

Textile fibers, examination of, 101, 158. 

Thickness, of cover-glass for non-adjust- 
able objectives, table, 14 ; of serial 
sections, 193. 

Tissues, arranging for sections, 191 ; fix- 
ing or hardening, 176, 183 ; washing 
apparatus for, 280. 

Tolles-Mayall mechanical stage, 67. 

Transections, 192. 

Transferring sections, 179. 

Transmitted light, 36. 



INDEX 



299 



Tray for slides, 187. 

Triplet, aplanatic, 7. 

Tripod, 7, 104 ; base for microscope, 86 ; 
as focusing glass, 209. 

Tube of microscope, Frontispiece. 

Tube-length, 13, 14,56, for cover-glass 
adjustment, 56, 57 ; importance of, 
56 ; microscopical, 13, 14 ; of various 
opticians, table, 14 ; and optical com- 
binations, 11S. 

Turn-table, 168. 

U— V— W— X 

Unadjustable objectives, 12. 

Under-correction, 5. 

Unit of measures, in micrometry, 112 ; of 
wave length, 143. 

Valuation of ocular micrometer, 114-116 ; 
table, in. 

Variable objective, 13. 

Varying magnification of compound mi- 
croscope, 109. 

Varving ocular micrometer valuation, 
118. 

Velocity under microscope, 98. 

Vertical, camera, 205-210, 225 : illumina- 
tor, 13, 238-239. 



Vials, preparation, 174 ; blocks for, 174. 

Virtual image, 5, 6, 9, 32 ; standard dis- 
tance at which measured, 109. 

Visibility with objectives, 20. 

Vision, double, 103, 105 ; microscopic, 21. 

Washing apparatus for tissues, 280. 

Waste bowl, 181. 

Water immersion objective, 16-19, 56; 
light utilized, 17 ; numerical aper- 
ture, 19 

Water, bath, 235, 255 ; for immersion ob- 
jectives and removal of, 56, 58. 

Wave length, designation of, 143 ; scale 
of, 142. 

Wax models, 274. 

Weights and measures, see 2d page of 
cover. 

Welsbach light, 37, 229. 

Wollaston's camera lucida, 107, 125. 

Work-room for photo-micrography, 224. 

Work-table, position, etc., 62. 

Working distance of microscope or ob- 
jective, 11, 34, 39-40. 

Writing diamond, 196. 

Xylene, 178, 200 ; balsam, 199 ; for re- 
moving oil, 179 ; removing from 
slides, 189. 

Xylol, German form of xylene, 178, 200. 



TABLE OF NATURAL SINES 
Compiled from Prof. G. W. f ones' Logarithmic Tables 



Mixutes. 



Degrees and Quarter Degrees up to 90° 



i 7 0.00029 

2 0.00058 

3 0.00087 

4 0.00116 

5 0.00145 
5 0.00175 

7 0.00204 

8 0.00233 

9 0.00262 
j 0.00291 

1 0.00320 

2 0.00349 

3 0.00378 

4 0.00407 
'5 0.00436 
'6 0.00465 

17 0.00495 

18 0.00524 

19 0.00553 

20 0.00582 

21 0.00611 

22 0.00640 
0.00669 

24 0.00698 

25 0.00727 

26 0.00756 

27 0.00785 

28 0.00814 

29 0.00844 

30 0.00873 

31 0.00902 
2 0.00931 

p3 0.00960 

34 0.00989 

35 0.01018 

36 0.01047 

37 0.01076 
8 0.01105 

39 0.01134 

40 0.0T164 

41 0.01193 

42 0.01222J 

43 0.01251 

44 0.01280I 

45 0.01309; 

46 0.01338 

47 0.01367 

48 0.01396 

49 0.01425 

50 0.01454 

51 0.01483: 

52 0.01513; 

53 0.01542 

54 0.01571 

55 0.01600 

56 0.01620 

57 0.01658 

58 0.01687 

59 0.01715 

60 0.0174- 



i°,I5' 

i,3° 
1.45 
2 

2,15 

2,30 

2,45 

3 

3,i5 

3,3o 

3,45 

4 

4,15 

4,30 

4,45 

5 

5,15 

5,30 

5,45 

6 

6,15 
6,30 

6,45 

7 

7,i5 

7,30 

7,45 

8 

8,15 
8.30 

8,45 
9 

9,i5 
9,30 
9,45 
10 

10,15 
10,30 

io,45 
11 

n,i5 
11,30 
ii,45 

12 

12,15 
12,30 

12,45 

13 

13,15 

13,30 

13,45 

14 

14,15 

14,30 

14,45 

15 

15,15 

15,30 

15,45 



0.01745 
0.02181 
0.02618 
0.03054 
0.03490 
0.03926 
0.04362 
0.04798 
0.05234 
0.05669 
0.06105 
0.06540 
0.06976 
0.0741 1 
0.07846 
0.08281 ; 
o.o87i6 ; 
0.09150 
0.09585 
o. 10019 
0.10453 j 
o. 10887 ' 
0.1 1320 

0.11754 

0.12187 
O.I2620J 

0.13053 
0.13485 
0.13917 
0.14349 

o. 14781 
o. 15212 

0.15643 

0.16074 

0.16505 
0.16935 

o. 17365 

0.17794 

0.18224 
0.18652 
0.1 908 1 

0.19509 
0.19937 

0.20364 
0.20791 
0.21218 
0.21644 
0.22070 

0.22495 

0.22920 

0.23345 
0.23769 

0.24192 
0.24615 

0.25038 

0.25460 
0.25882 

0.26303 

0.26724 
0,27144! 



16 , 


0.27564:31°, 


0. 


i6°,i5 


'o.27983!3i°,i5 


'0. 


16,30 


0.28402 31,30 


0. 


i6,45 


0.2882031,45 


0. 


17 


0.2923732 


0. 


17,15 


0.2965432,15 


0. 


17,30 


0.30071 32,30 


0. 


17,45 


0.3048632,45 


0. 


18 


0.3090233 


0. 


18,15 


0.3131633,15 


0. 


18,30 


o.3i730'33,30 


0. 


iS,45 


0.3214433,45 


0. 


19 


o.32557 34 


0. 


19,15 


0.32969 34, 1 5 


0. 


19,30 


o.3338i 34,30 


0. 


19,45 


o.3379 2 34,45 


0. 


20 


0.3420235 


0. 


20,15 


0.3461235,15 


0. 


20,30 


0.35021 35,30 


0. 


20,45 


o.35429 35,45 


0. 


21 


o-35837 l 3 6 


0. 


21,15 


0.3624436,15 


0. 


21,30 


0.3665036,30 


0. 


21,45 


0.3705636,45 


0. 


22 


o.3746ii37 


0. 


22,15 


o.3786537,i5 


0. 


22,30 


0.3826837,30 


0. 


22,45 


0.38671^7,45 


0. 


23 


0.39073 38 


0. 


23,15 


0.3947438,15 


0. 


23,30 


0.39875 38,30 





23,45 


0.40275,38,45 


0. 


24 


0.4067439 


0. 


24,15 


0.4107239,15 


0. 


24,30 


0.4146939,30 


0. 


24,45 


0.4186639,45 


0. 


25 


0.4226240 


0. 


25,15 


0.4265740,15 


0. 


25,30 


0.4305140,30 


0. 


25,45 


o.43445 40,45 


0. 


26 


0.43837 4 1 


0. 


26,15 


0.4422941,15 


0. 


26,30 


0.4462041,30 


0. 


26,45 


0.45010 41,45 


0. 


27 


0-45399 4 2 


0. 


27,15 


0.4578742,15 


0. 


27,30 


0.4617542,30 


0. 


27,45 


0.4656142,45 


0. 


28 


0.46947 43 


0. 


28,15 


0.4733243,15 


0. 


28,30 


0.4771643,30 


0. 


28,45 


0.4809943,45 


0. 


29 


0.4848144 


0. 


29,15 


0.4886244,15 


0. 


29,30 


0.49242 44,30 


0. 


29,45 


0.4962244,45 


0. 


30 


0.5000045 


0. 


30,15 


0.50377 45,15 


0. 


30,30 


0.50754 45,30 


0. 


30,45 


0.5112945,45 


0. 



5150446°, 

5i877; 4 6°,i5' 
5225046,30 

5262146,45 

52992i47 
5336147,15 

5373047,30 

54097 47,45 

54464 148 

5482948,15 

5519448,30 

5555748,45 

559J949 

56280:49,15 

56641(49,30 

5700049,45 

5735850 

57715 50,15 
58070150,30 
58425^0,45 

58779'5i 

59131151,15 

59482.51,30 

■59832,51.45 

.60182:52 

60529152,15 

6087652, 30 

6122252,45 

6156653 

61909^3,15 
62251 53,30 
62592 53,45 
62932 54 
.6327154,15 
.63608 54,30 
,63944:54,45 

•64279'55 
.6461255,15 

•64945 55,30 
• 65276 55,45 
.6560656 

.65935 56,15 
,66262 56,30 

,6658856,45 

■ 6691357 

.67237:57,15 

67559 , 57,3o 

■ 67880 1 5 7, 45 
,6820058 
.6851858,15 
.6883558,30 
,69151 58,45 
.69466 59 

69779 59,15 
.70091 59,30 

70401 59,45 

.70711160 

,71019:60,15 

71325160,30 

71630160,45 



0.71934 
0.72236 

0.72537; 
0.72837' 

o^^ 
o.73432i 
0.73728 
0.74022 

o.743i4| 

0.74606 

o. 74896 

0.75184 

o.7547 

0.75756, 

0.76041; 

0.76323! 
0.76604: 
0.76884, 
0.77162J 

0-77439J 

o.777i5 
0.7798S 
0.78261 
0.78532 
0.78801 
o. 79069 

o.79335 
o. 79600 
0.79864 
0.80125 

0.80386 

o. 80644 
o. 80902 
0.81157 
0.81412 
0.81664 

0.81915 

0.82165 
0.82413 
0.82659 
0.82904 

0.83147 
0.83389 

0.83629 

0.83867 

0.84104 

0.84339 
0.84573 

0,84805 

0.85035 

0.85264 

0.85491 
0.85717 
0.85941 
0.86163 
0.86384 

0.86603 

0.86820 

0.87036 
0.87250 



6i°, 

6i°,i5 / 
61,30 

6i,45 

62 

62,15 

62,30 

62,45 

63 

63,15 

63,30 

63,45 

64 

64,15 

64,30 

64,45 

65 

65,15 

65,30 

65,45 

66 

66,15 

66,30 

66,45 

j6 7 

167,15 

67,30 

67,45 

68 

68,15 

68,30 

68,45 

69 

69,15 

69,30 

69,45 

7o 

7o,i5 

70,30 

7o,45 

7i 

7i,i5 

71,30 

7i,45 

72 

72,15 

72,30 

72,45 

73 

73,15 

73,3o 

73,45 

74 

74,15 

74,30 

74,45 

75 

75,15 

75,30 

75,45 



0.87462 
0.87673 
0.87882 
0.88089 
0.88295 
0.88499: 
0.88701 
0.88902 
0.89101 
0.89298 
0.89493 
0.89687 
0.89879 
0.90070 
0.90259 
o. 90446 
o. 9063 1 
0.90814 
o. 90996 
0.91176 

0.91355' 
0.91531 

0.91706 
0.91879 
0.92050 
0.92220 

0.92388 
0.92554 

0.92718 

0.92881 
0.93042 

0.93201 

0.93358 
0.93514 
0.93667 

0.93819 

0.93969 

0.94118 
0.94264 

0.94409 
0.94552 
0.94693 
0.94832 

0.94970 

0.95106 
0.95240 
0.95372 
0.95502 
0.95630 
0.95757 

0.95S82 
0.96005 
0.96126 
0.96246 
0.96363 

0.96479 

0.96593 
0.96705 
0.96815 
0.96923 



76°, o. 
76°,i5 / o. 
76,30 o. 
76,45 o. 

77 o. 
77,15 o. 
77,3o o. 
77,45 o. 

78 o. 
78,15 o. 
78,30 o. 
78,45 
79 

79,15 
J79,3° 
79,45 
!8o 
80,15 

:80,30 

1 80, 45 

81 

|8i,i5 
81,30 

8i,45 
82 

82,15 
82,30 

82,45 

83 

83,15 

83,30 

83,45 

84 

84,^5 

84,30 

84,45 

85 

85,15 

85,30 

85,45 

86 

86,15 

86,30 

86,45 

87 

87,15 

87,30 

87,45 

88 

88,15 

88,30 

88,45 



89,15 
89,30 
89,45 
90 



97030 
97134 
97237 
97338 
97437 
97534 
97630 
97723 
978i5 
97905 
97992 
98079 
98163 
98245 
98325 
98404 
98481 
98556 
98629 
98700 
98769 
98836 
98902 
98965 
99027 
99087 
99144 
99200 
99255 
99307 
99357 
99406 
99452 
99497 
99540 
9958o 
99619 

99657 
99692 

99725 
99756 
99786 
99813 
99839 
.99863 
99SS5 
99905 
99923 
99939 
9V953 
99966 
99976 
99985 
99991 
99996 

99999 
00000 










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